Oxygenation-linked subunit interactions in human hemoglobin: experimental studies on the concentration dependence of oxygenation curves

An experimental study on the concentration dependence of oxygenation curves for human hemoglobin has been carried out between 4 X 10(-8) M heme and 5 X 10(-4) M heme in 0.1 M tris(hydroxymehtyl)aminomethane hydrochloride, 0.1 M NaCl, 1 mM disodium ethylenediaminetetraacetic acid, pH 7.4, 21.5 degrees C. With decreasing hemoglobin concentration the curves show pronounced shifts in position and shape, consistent with dissociation of tetrameric hemoglobin into dimeric species of high affinity and low cooperativity. Combination of these data with independently determined values of dissociation constants for unliganded and fully liganded hemoglobin permits a resolution of the seven parameters necessary to define the linked binding and subunit association processes. The total oxygenation-linked subunit dissociation energy (6.34 kcal) was resolved into intersubunit contact energy changes between alphabeta dimers in tetrameric hemoglobin which accompany binding of the first, middle two, and last oxygen molecules. The resolution is accurate to within approximately +/-0.3 kcal. To within this limit the isolated dimers are found to bind oxygen noncooperatively and with the same affinity as isolated alpha and beta chains. Equally good fits to the data are obtained when dimers are slightly anticooperative. At least three major energetic states are apparently assumed by hemoglobin tetramers, with respect to the alpha1beta2 contact region, corresponding to (a) unliganded, (b) singly liganded, (c) triply and quadruply liganded species. The results do not establish whether these states may be assumed by a single molecule, or whether they arise as averages over a distrubution of conformational states. They do provide unequivocal evidence against a concerted transition at any particular binding step in a system with only two energetic states of tetramer (i.e., an all or none switchover between T and R states at a particular binding step).     

Quaternary structure regulates hemin dissociation from human hemoglobin

Rate constants for hemin dissociation from the alpha and beta subunits of native and recombinant human hemoglobins were measured as a function of protein concentration at pH 7.0, 37 degrees C, using H64Y/V68F apomyoglobin as a hemin acceptor reagent. Hemin dissociation rates were also measured for native isolated alpha and beta chains and for recombinant hemoglobin tetramers stabilized by alpha subunit fusion. The rate constant for hemin dissociation from beta subunits in native hemoglobin increases from 1.5 h-1 in tetramers at high protein concentration to 15 h-1 in dimers at low concentrations. The rate of hemin dissociation from alpha subunits in native hemoglobin is significantly smaller (0.3-0.6 h-1) and shows little dependence on protein concentration. Recombinant hemoglobins containing a fused di-alpha subunit remain tetrameric under all concentrations and show rates of hemin loss similar to those observed for wild-type and native hemoglobin at high protein concentration. Rates of hemin dissociation from monomeric alpha and beta chains are much greater, 12 and 40 h-1, respectively, at pH 7, 37 degrees C. Aggregation of monomers to form alpha1beta1 dimers greatly stabilizes bound hemin in alpha chains, decreasing its rate of hemin loss approximately 20-fold. In contrast, dimer formation has little stabilizing effect on hemin binding to beta subunits. A significant reduction in the rate of hemin loss from beta subunits does occur after formation of the alpha1beta2 interface in tetrameric hemoglobin. These results suggest that native human hemoglobin may have evolved to lose heme rapidly after red cell lysis, allowing the prosthetic group to be removed by serum albumin and apohemopexin.     

Nanosecond time-resolved absorption studies of human oxyhemoglobin photolysis intermediates

The time-resolved spectra of photoproducts from ligand photodissociation of oxyhemoglobin are measured in the Soret spectral region for times from 10 ns to 320 microseconds after laser photolysis. Four processes are detected at a heme concentration of 80 microM: a 38-ns geminate recombination, a 137-ns tertiary relaxation, and two bimolecular processes for rebinding of molecular oxygen. The pseudo-first-order rate constants for rebinding to the alpha and beta subunits of hemoglobin are 3.2 x 10(4) s-1 (31 microseconds lifetime) and 9.4 x 10(4) s-1 (11 microseconds lifetime), respectively. The significance of kinetic measurements made at different heme concentrations is discussed in terms of the equilibrium compositions of hemoglobin tetramer and dimer mixtures. The rebinding rate constants for alpha and beta chains are observed to be about two times slower in the dimer than in the tetramer, a finding that appears to support the observation of quaternary enhancement in equilibrium ligand binding by hemoglobin tetramers.     

Oxygenation-linked subunit interactions in human hemoglobin: analysis of linkage functions for constituent energy terms

Resolution of the linkage functions between oxygenation and subunit association-dissociation equilibria in human hemoglobin into the constituent microscopic terms has been explored by numerical simulation and least-squares analysis. The correlation properties between parameters has been studied using several choices of parameter sets in order to optimize resolution. It is found that, with currently available levels of experimental precision and ranges of variables, neither linkage function can provide sufficient resolution of all the desired energy terms. The most difficult quantities to resolve always include the dimer-tetramer association constant for unliganded hemoglobin and the oxygen binding constants to alphabeta dimers. A feasible experimental strategy for overcoming these difficulties lies in independent determination of the dimer-tetramer association constants for unliganded and fully oxygenated hemoglobin. These constants, in combination with the median lignad concentration, provide an estimate of the energy for total oxygenation of tetramers which is essentially independent of the other constituent energies. It is shown that if these separately determinable parameters are fixed, the remaining terms may be estimated to good accuracy using data which represents either linkage function. In general it is desirable to combine information from both types of experimental quantities. A previous paper (Mills, F.C., Johnson, M.L., and Ackers, G.K. (1976), Biochemestry, 15, the preceding paper in this issue) describes the experimental implementation of this strategy.     

Asymmetric cyanomet valency hybrid hemoglobin, (alpha+CN-beta+CN-)(alpha beta): the issue of valency exchange

A new framework for hemoglobin cooperativity was proposed by Ackers and colleagues on the basis of the hyper thermodynamic stability and deoxy (T) quaternary structure of one of diliganded deoxy-cyanomet hybrid hemoglobins, (alpha+CN-beta+CN-)(alpha beta), studied by hybridization of the equimolar mixture of deoxyhemoglobin and cyanomethemoglobin through a long (70-100 h) dimer exchange reaction [Daugherty et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1110-1114]. Recently, we reported that the published hyperstability of (alpha+CN-beta+CN-)(alpha beta) is incorrect due to the occurrence of valency exchange between the heme sites of both parental hemoglobins during the long deoxy incubation [Shibayama et al. (1997) Biochemistry 36, 4375-4381]. We also noted a difficulty in maintaining both anaerobicity and excess free cyanide of the sample during the long incubation, which led to formation of cyanide-unbound aqometheme in the original deoxyhemoglobin resulting from the electron transfer to cyanometheme. This paper is a response to a recent argument against our work [Ackers et al. (1997) Biochemistry 36, 10822-10829]. Ackers et al. have claimed that no appreciable formation of aqomethemoglobin with their methods ensures their sample integrity, based on a supposition that our observed valency exchange may have occurred via aqometheme. In this paper, however, we demonstrate that appreciable (>27%) valency exchange really occurs between deoxy and cyanometheme sites during 72 h incubation under conditions where both anaerobicity and excess free cyanide of the sample solution are maintained by a continuous flow of humidified N2 with HCN. This confirms our view that previous experimental data on (alpha+CN-beta+CN-)(alpha beta) obtained by the long incubations should be subject to reexamination while our earlier estimation of a lower limit of free energy of (alpha+CN-beta+CN-)(alpha beta) (i.e., >/= -10.1 kcal/mol) by a rapid method (35 min) is still valid. We also suggest a possibility that the T quaternary structure of (alpha+CN-beta+CN-)(alpha beta) assigned by Ackers and colleagues using the long incubations is an artifact arising from the valency exchange. These results suggest that the putative mechanistic picture for hemoglobin cooperativity inferred from studies on deoxy-cyanomet hybrids is without foundation.     

Tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation rates

One of the main difficulties with blood substitutes based on hemoglobin (Hb) solutions is the auto-oxidation of the hemes, a problem aggravated by the dimerization of Hb tetramers. We have employed a method to study the oxyHb tetramer-dimer equilibrium based on the rate of auto-oxidation as a function of protein concentration. The 16-fold difference in dimer and tetramer auto-oxidation rates (in 20 mM phosphate buffer at pH 7.0, 37 degrees C) was exploited to determine the fraction dimer. The results show a transition of the auto-oxidation rate from low to high protein concentrations, allowing the determination of the tetramer-dimer dissociation coefficient K4,2 = [Dimer] 2/[Tetramer]. A 14-fold increase in K4,2 was observed for addition of 10 mM of the allosteric effector inositol hexaphosphate (IHP). Recombinant hemoglobins (rHb) were genetically engineered to obtain Hb with a lower oxygen affinity than native Hb (Hb A). The rHb alpha2beta2 [(C7) F41Y/(G4) N102Y] shows a fivefold increase in K4,2 at pH 7.0, 37 degrees C. An atmosphere of pure oxygen is necessary in this case to insure fully oxygenated Hb. When this condition is satisfied, this method provides an efficient technique to characterize both the tetramer-dimer equilibrium and the auto-oxidation rates of various oxyHb. For low oxygen affinity Hb equilibrated under air, the presence of deoxy subunits accelerates the auto-oxidation. Although a full analysis is complicated, the auto-oxidation studies for air equilibrated samples are more relevant to the development of a blood substitute based on Hb solutions. The double mutants, rHb alpha2beta2 [(C7) F41Y/(G4) N102A] and rHb alpha2beta2 [(C7) F41Y/(E10) K66T], show a lower oxygen affinity and a higher rate of oxidation than Hb A. Simulations of the auto-oxidation rate versus Hb concentration indicate that very high protein concentrations are required to observe the tetramer auto-oxidation rate. Because the dimers oxidize much more rapidly, even a small fraction dimer will influence the observed oxidation rate.     

Preparation and kinetic characterization of a series of betaW37 variants of human hemoglobin A: evidence for high-affinity T quaternary structures

Four variants of human beta globin in which the Trp at position 37 has been replaced with a Tyr, Ala, Gly, or Glu have been expressed in Escherichia coli. These globins have been combined with normal human alpha chains and heme to form tetrameric hemoglobin molecules. A technique for the preparation of alpha chain dimers, which are cross-linked between their alpha99 lysine residues, has been developed, and these alpha dimers were combined with two of the beta globins, betaW37G and betaW37E, to form the corresponding cross-linked variants. The kinetics of CO binding to the deoxygenated derivatives following rapid mixing and of CO rebinding following flash photolysis have been examined as functions of pH in the presence and absence of the organic phosphate inositol hexaphosphate, IHP. The kinetic measurements indicate that replacement of the tryptophan with other residues destabilizes the hemoglobin tetramer, resulting in considerable dissociation of even the deoxygenated hemoglobins into alphabeta dimers at micromolar protein concentrations. Substitutions at beta37 also alter the properties of the deoxygenated hemoglobin tetramer. The alteration of the functional properties of the T states of these variants as well as the tendency of the deoxygenated derivatives to dissociate into alphabeta dimers increases in the order HbA < betaW37Y < betaW37A < betaW37G < betaW37E. Stabilizing the betaW37G or betaW37E tetramers by addition of IHP or by cross-linking does not restore the normal functional properties of the T state. Measurements of the geminate rebinding of CO establish a kinetic difference between the normal R state tetramer and the alphabeta dimer consistent with quaternary enhancement, the greater affinity of oxygen for the R state tetramer than for the alphabeta dimer. Kinetics of geminate rebinding also suggest that quaternary enhancement may be altered by substitutions at the beta37 position.     

Discussion of “the influence of chemical equilibrium on fluid-solid reaction rates and the falsification of activation energy”

In a recent article, Sohn^[1] tried to establish, by way of demonstrating the importance of thermodynamics in kinetic analyses, that fluid-solid reactions (forming no solid product layer) with small equilibrium constants tend to be kinetically controlled by mass transfer, displaying falsified apparent activation energies. Admitting the thermodynamic involvement in such analyses, the present discussion tries to counterestablish through an example that those reactions may as well tend to be chemically controlled or follow a mixed control kinetics, in which cases no such falsification occurs. H.Y. SOHN: Metall. Mater. Trans. B, 2004, vol. 35B, pp. 121–31. An erratum to this article is available at .     

Identifying the conformational state of bi-liganded haemoglobin

While most researchers agree on the global features of cooperative ligand binding to haemoglobin (Hb), the internal mechanisms remain open to debate. This is not due to inaccurate measurements, but is rather a consequence of the cooperative ligand binding that decreases the equilibrium populations of the partially liganded states and makes observation of the transitions between these substates more difficult. For example, the equilibrium population of the doubly liganded tetramers is typically less than 5% of the total Hb. As a result many models with widely varying mechanisms may fit the oxygen equilibrium curve, but may not be consistent with observations of other parameters, such as ligand-binding kinetics or subunit association equilibria. The wide range of methods and models has led to divergent conclusions about the properties of specific substates. One notable debate concerns the properties of the doubly liganded forms. The simple two-state model predicts a shift in the allosteric equilibrium based on the number of ligands bound, but not on their distribution within the tetramer. From studies of dimer-tetramer equilibria of various pure and hybrid forms, it was concluded that a tetramer with two ligands bound on the same alpha beta dimer (species 21, an asymmetric hybrid) shows an enhanced tetramer stability, similar to singly liganded Hb, relative to the other three types of doubly liganded tetramers which resemble the triply liganded forms [Ackers et al. (1992). Science 255: 54-63]. The implications of this model and the relevant experiments will be reviewed here.     

The TATA-binding protein from Saccharomyces cerevisiae oligomerizes in solution at micromolar concentrations to form tetramers and octamers

Equilibrium analytical ultracentrifugation has been used to determine the stoichiometry and energetics of the self-assembly of the TATA-binding protein of Saccharomyces cerevisiae at 30 degreesC, in buffers ranging in salt concentration from 60 mM KCl to 1 M KCl. The data are consistent with a sequential association model in which monomers are in equilibrium with tetramers and octamers at protein concentrations above 2.6 microM. Association is highly cooperative, with octamer formation favored by approximately 7 kcal/mol over tetramers. At high [KCl], the concentration of tetramers becomes negligible and the data are best described by a monomer-octamer reaction mechanism. The equilibrium association constants for both monomer <--> tetramer and tetramer <--> octamer reactions change with [KCl] in a biphasic manner, decreasing with increasing [KCl] from 60 mM to 300 mM, and increasing with increasing [KCl] from 300 mM to 1 M. At low [KCl], approximately 3 mole equivalents of ions are released at each association step, while at high [KCl], approximately 3 mole equivalents of ions are taken up at each association step. These results suggest that there is a salt concentration-dependent change in the assembly mechanism, and that the mechanistic switch takes place near 300 mM KCl. The possibility that this self-association reaction may play a role in the activity of the TATA-binding protein in vivo is discussed.     

Arterial blood pressure responses to cell-free hemoglobin solutions and the reaction with nitric oxide

Changes in mean arterial pressure were monitored in rats following 50% isovolemic exchange transfusion with solutions of chemically modified hemoglobins. Blood pressure responses fall into three categories: 1) an immediate and sustained increase, 2) an immediate yet transient increase, or 3) no significant change either during or subsequent to exchange transfusion. The reactivities of these hemoglobins with nitric monoxide (.NO) were measured to test the hypothesis that different blood pressure responses to these solutions result from differences in .NO scavenging reactions. All hemoglobins studied exhibited a value of 30 microM-1 s-1 for both .NO bimolecular association rate constants and the rate constants for .NO-induced oxidation in vitro. Only the .NO dissociation rate constants and, thus, the equilibrium dissociation constants varied. Values of equilibrium dissociation constants ranged from 2 to 14 pM and varied inversely with vasopressor response. Hemoglobin solutions that exhibited either transient or no significant increase in blood pressure showed tighter .NO binding affinities than hemoglobin solutions that exhibited sustained increases. These results suggest that blood pressure increases observed upon exchange transfusion with cell-free hemoglobin solutions can not be the result of .NO scavenging reactions at the heme, but rather must be due to alternative physiologic mechanisms.     

Oxygen equilibrium properties of chromium (III)-iron (II) hybrid hemoglobins

Cr(III)-Fe(II) hybrid hemoglobins, alpha 2(Cr) beta 2(Fe) and alpha 2(Fe) beta 2(Cr), in which hemes in either the alpha- or beta-subunits were substituted with chromium(III) protoporphyrin IX (Cr(III)(PPIX), were prepared and characterized by oxygen equilibrium measurements. Because Cr(III)PPIX binds neither oxygen molecules nor carbon monoxide, the oxygen equilibrium properties of Fe(II) subunits within these hybrids can be analyzed by a two-step oxygen equilibrium scheme. The oxygen equilibrium constants for both hybrids at the second oxygenation step agree with those for human adult hemoglobin at the last oxygenation step (at pH 6.5-8.4 with an without inositol hexaphosphate at 25 degrees C). The similarity between the effects of the Cr(III)PPIX and each subunits' oxygeme on the oxygen equilibrium properties of the counterpart Fe(II) subunits within hemoglobin indicate the utility of Cr(III)PPIX as a model for a permanently oxygenated heme within the hemoglobin molecule. We found that Cr(III)-Fe(II) hybrid hemoglobins have several advantages over cyanomet valency hybrid hemoglobins, which have been frequently used as a model system for partially oxygenated hemoglobins. In contrast to cyanomet heme, Cr(III)PPIX within hemoglobin is not subject to reduction with dithionite or enzymatic reduction systems. Therefore, we could obtain more accurate and reasonable oxygen equilibrium curves of Cr(III)-Fe(II) hybrids in the presence of an enzymatic reduction system, and we could obtain single crystals of deoxy-alpha 2(Cr) beta 2(Fe) when grown in low salt solution in the presence of polyethylene glycol 1000 and 50 mM dithionite.     

On the molecular mechanism of chloroquine's antimalarial action

Chloroquine is thought to exert its antimalarial effect by preventing the polymerization of toxic heme released during proteolysis of hemoglobin in the Plasmodium digestive vacuole. The mechanism of this blockade has not been established. We incubated cultured parasites with subinhibitory doses of [3H]chloroquine and [3H] quinidine. These [3H]quinoline compounds became associated with hemozoin as assessed by electron microscope autoradiography and subcellular fractionation. In vitro, binding of [3H]quinoline inhibitors to the hemozoin chain depended on the addition of heme substrate. These data counter previous conclusions regarding the lack of quinoline association with hemozoin, explain the exaggerated accumulation of quinolines in the plasmodium digestive vacuole, and suggest that a quinoline heme complex incorporates into the growing polymer to terminate chain extension, blocking further sequestration of toxic heme.     

Thermodynamic cycle between DNA and RNA constituents for conformation of the sugar ring from nuclear magnetic resonance study

The effect of a structural change of ribose to deoxyribose, by replacement of 2'-OH by 2'-H, on the conformational equilibrium of the sugar ring is described in terms of one thermodynamic cycle. The method is based on the observation that conformational correlations of the sugar ring--side chain ensemble in DNA and RNA components show one general pattern, reflecting an intrinsic physical property of this ensemble. The pattern determines a choice of model systems to study. The systems consist of pairs of DNA and RNA components, nucleosides and nucleotides in aqueous solution, where all conformational factors are fully controlled. This approach allowed us to describe the thermodynamic cycle and measure its fundamental parameters, equilibrium constants and free energy differences, delta delta G, from a nuclear magnetic resonance study. The delta delta G values as determined for pairs of ribo- and deoxyribo-nucleosides in classes of syn-constrained and anti-preferred models, are comparable and lie in a narrow range, delta delta G = 1.7 +/- 0.1 [kJ/mol]. For pairs of ribo- and deoxyribo-nucleotides, the delta delta G values also lie in narrow ranges, delta delta G = 1.7 +/- 0.1 [kJ/mol] for 5'-phosphate nucleotides and delta delta G = 1.9 +/- 0.1 [kJ/mol] for 3'-phosphate nucleotides, i.e. similar to those observed for nucleosides. The measured quantity, delta delta G, is generally observed in a relatively narrow range, delta delta G = 1.75 +/- 0.15 [kJ/mol], irrespective of the class of the model system. This quantity represents a "pure" constant contribution, pe one sugar moiety, as a "driving force" for the N-->S shift in the sugar ring conformational equilibrium, when one compares RNA and DNA. This important thermodynamic quantity, delta delta G, has not hitherto been determined for nucleic acids. Ultimately the delta delta G quantity is revealed in the tendency to adopt S(C2'endo) sugar puckering domain by the majority of DNA structures, whereas RNA generally adopt an N(C3'endo) puckering domain. A possible biological significance of the delta delta G quantity may include evolutionary aspects of nucleic acids.     

Electron paramagnetic resonance and oxygen binding studies of alpha-Nitrosyl hemoglobin. A novel oxygen carrier having no-assisted allosteric functions

alpha-Nitrosyl hemoglobin, alpha(Fe-NO)2beta(Fe)2, which is frequently observed upon reaction of deoxy hemoglobin with limited quantities of NO in vitro as well as in vivo, has been synthetically prepared, and its reaction with O2 has been investigation by EPR and thermodynamic equilibrium measurements. alpha-Nitrosyl hemoglobin is relatively stable under aerobic conditions and undergoes reversible O2 binding at the heme sites of its beta-subunits. Its O2 binding is coupled to the structural/functional transition between T- (low affinity extreme) and R- (high affinity) states. This transition is linked to the reversible cleavage of the heme Fe-proximal His bonds in the alpha(Fe-NO) subunits and is sensitive to allosteric effectors, such as protons, 2,3-biphosphoglycerate, and inositol hexaphosphate. In fact, alpha(Fe-NO)2beta(Fe)2 is exceptionally sensitive to protons, as it exhibits a highly enhanced Bohr effect. The total Bohr effect of alpha-nitrosyl hemoglobin is comparable to that of normal hemoglobin, despite the fact that the oxygenation process involves only two ligation steps. All of these structural and functional evidences have been further confirmed by examining the reactivity of the sulfhydryl group of the Cysbeta93 toward 4, 4'-dipyridyl disulfide of several alpha-nitrosyl hemoglobin derivatives over a wide pH range, as a probe for quaternary structure. Despite the halved O2-carrying capacity, alpha-nitrosyl hemoglobin is fully functional (cooperative and allosterically sensitive) and could represent a versatile low affinity O2 carrier with improved features that could deliver O2 to tissues effectively even after NO is sequestered at the heme sites of the alpha-subunits. It is concluded that the NO bound to the heme sites of the alpha-subunits of hemoglobin acts as a negative allosteric effector of Hb and thus might play a role in O2/CO2 transport in the blood under physiological conditions.     

REM sleep behavior disorder and degenerative dementia: an association likely reflecting Lewy body disease

It is commonly believed that MgATP2- is the substrate of F1-ATPases and ATP4- acts as a competitive inhibitor. However, the velocity equation for such competitive inhibition is equivalent to that for a rapid equilibrium ordered binding mechanism in which ATP4- adds first and the binding of Mg2+ is dependent on the formation of the E x ATP4- complex. According to this ordered-binding model, solution formed MgATP2- is not recognized by the ATPase as a direct substrate, and the high-affinity binding of Mg2+ to the E x ATP4- complex is the key reaction towards the formation of the ternary complex. These models (and others) were tested with an F1- ATPase, isolated from Halobacterium saccharovorum, by evaluating the rate of ATP hydrolysis as a function of free [ATP4-] or free [Mg2+]. The rates were asymmetrical with respect to increasing [ATP4-] versus increasing [Mg2+]. For the ordered-binding alternative, a series of apparent dissociation constants were obtained for ATP4-(K(A)aPP), which decreased as [Mg2+] increased. From this family of K(A)aPP the true K(A) was retrieved by extrapolation to [Mg2+] = 0 and was found to be 0.2 mM. The dissociation constants for Mg2+, established from these experiments, were also apparent (K(B)aPP) and dependent on [ATP4-] as well as on the pH. The actual K(B) was established from a series of K(B)aPP by extrapolating to [ATP4-] = infinity and to the absence of competing protons, and was found to be 0.0041 mM. The pKa of the protonable group for Mg2+ binding is 8.2. For the competitive inhibition alternative, rearrangement of the constants and fitting to the velocity equation gave an actual binding constant for MgATP2- (K(EAB)) of 0.0016 mM and for ATP4- (K(EA)) of 0.2 mM. Decision between the two models has far-reaching mechanistic implications. In the competitive inhibition model MgATP2- binds with high affinity, but Mg2+ cannot bind once the E x ATP4- complex is formed, while in the ordered-binding model binding of Mg2+ requires that ATP4- adds first. The steric constraints evident in the diffraction structure of the ATP binding site in the bovine mitochondrial F-ATPase [Abrahams, J. P., Leslie, A. G. W., Lutter, R. & Walker, J. E. (1994) Nature 370, 621-628] tend to favor the ordered-binding model, but the final decision as to which kinetic model is valid has to be from further structural studies. If the ordered-binding model gains more experimental support, a revision of the current concepts of unisite catalysis and negative cooperativity of nucleotide binding will be necessary.     

Solution structure of human neuropeptide Y

The three-dimensional structure of synthetic human neuropeptide Y in aqueous solution at pH 3.2 and 37 degrees C was determined from two-dimensional 1H NMR data recorded at 600 MHz. A restraint set consisting of 440 interproton distance restraints inferred from NOEs and 11 backbone and 4 side-chain dihedral angle restraints derived from spin-spin coupling constants was used as input for distance geometry calculations on DIANA and simulated annealing and restrained energy minimization in X-PLOR. The final set of 26 structures is well defined in the region of residues 11-36, with a mean pairwise rmsd of 0.51 A for the backbone heavy atoms (N, C alpha and C) and 1.34 A for all heavy atoms. Residues 13-36 form an amphipathic alpha-helix. The N-terminal 10 residues are poorly defined relative to the helical region, although some elements of local structure are apparent. At least one of the three prolines in the N-terminal region co-exists in both cis and trans conformations. An additional set of 24 distances was interpreted as intermolecular distances within a dimer. A combination of distance geometry and restrained simulated annealing yielded a model of the dimer having antiparallel packing of two helical units, whose hydrophobic faces form a well-defined core. Sedimentation equilibrium experiments confirm the observation that neuropeptide Y associates to form dimers and higher aggregates under the conditions of the NMR experiments. Our results therefore support the structural features reported for porcine neuropeptide Y [Cowley, D.J. et al. (1992) Eur. J. Biochem., 205, 1099-1106] rather than the 'aPP' fold described previously for human neuropeptide Y [Darbon, H. et al. (1992) Eur. J. Biochem., 209, 765-771].     

The behaviour of chromium in reduced slag-metal systems

The equilibrium between slag and metal in the CaO-SiO₂-Al₂O₃-CrO_x-FeO/Fe-Cr-Si system was investigated and the results expressed in terms of the apparent equilibrium constants for the Cr/FeO and Cr/SiO₂ equilibria. The effect of slag composition and temperature on the apparent equilibrium constants is described. The predominant form of chromium in the slags was CrO and the thermodynamic properties of CrO in the slag are discussed. The equilibrium results are applied to the stainless steelmaking system in order to show the effect of slag composition, silicon content of metal and temperature on the equilibrium chromium content of the slag.     

Role of the strictly conserved tryptophan-25 residue in the stabilization of the structure and in the ligand binding properties of the kringle 2 domain of tissue-type plasminogen activator

The involvement of the strictly conserved tryptophan-25 (W25) residue in the structural stability and omega-amino acid ligand binding properties of the recombinant (r) kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) has been investigated. Two conservative mutants were constructed and expressed that contained W25-->F and W25-->Y substitutions. The binding (dissociation) constants (Kd) for three ligands, viz., 6-aminohexanoic acid (EACA), 7-aminoheptanoic acid (7-AHpA), and L-lysine (Lys), to these polypeptides were determined by intrinsic fluorescence titrations. In the case of r-[K2tPA/W25F], the Kd values for these ligands were found to be 37, 16, and 89 microM for EACA, 7-AHpA, and Lys, respectively. For r-[K2tPA/W25Y], the Kd values for these same ligands were 64, 9, and 115 microM, respectively. The wild-type (wt) kringle domain possessed Kd values of 43, 6, and 85 microM for EACA, 7-AHpA, and Lys, respectively. The effect of these mutations on the stability of the r-[K2tPA] domain has been examined by differential scanning colorimetry. The temperature of maximum heat capacity (Tm) of wt-r-[K2tPA] (75.6 degrees C) was dramatically reduced to 50.8 and 58.0 degrees C for r-[K2tPA/W25F] and r-[K2tPA/W25Y], respectively. In the presence of EACA, the Tm values were increased to 86.1, 61.7, and 68.7 degrees C, respectively, indicating that EACA does interact with the r-[K2tPA] mutants and stabilizes their native conformations, similar to the case with wt-r-[K2tPA].(ABSTRACT TRUNCATED AT 250 WORDS)     

坩埚实验中合金结构钢液 渣反应热力学

 通过实验室坩埚实验,探讨了1 873 K下合金结构钢液、渣、夹杂物之间的热力学关系。首先通过预平衡实验,确定了钢 渣反应达到平衡所需时间为90 min。随后,进行了7炉平衡实验,探讨了平衡实验中钢液、夹杂物、渣三者间的关系。结果表明,1 873 K下,钢液与炉渣反应90 min后,钢中夹杂物主要为2 μm左右的细小球形MgO·Al2O3尖晶石夹杂物。钢 渣反应平衡时,镁铝尖晶石夹杂物中log(XMgO/XAl2O3)分别与钢液中log[a[Mg]/(a2[Al]·a2[O])]、渣中log(aMgO/aAl2O3)线性关系良好,斜率接近于1。