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• W1969361812 abstract "The O2 equilibria of human adult hemoglobin have been measured in a wide range of solution conditions in the presence and absence of various allosteric effectors in order to determine how far hemoglobin can modulate its O2 affinity. The O2 affinity, cooperative behavior, and the Bohr effect of hemoglobin are modulated principally by tertiary structural changes, which are induced by its interactions with heterotropic allosteric effectors. In their absence, hemoglobin is a high affinity, moderately cooperative O2 carrier of limited functional flexibility, the behaviors of which are regulated by the homotropic, O2-linked T/R quaternary structural transition of the Monod-Wyman-Changeux/Perutz model. However, the interactions with allosteric effectors provide such “inert” hemoglobin unprecedented magnitudes of functional diversities not only of physiological relevance but also of extreme nature, by which hemoglobin can behave energetically beyond what can be explained by the Monod-Wyman-Changeux/Perutz model. Thus, the heterotropic effector-linked tertiary structural changes rather than the homotropic ligation-linked T/R quaternary structural transition are energetically more significant and primarily responsible for modulation of functions of hemoglobin. The O2 equilibria of human adult hemoglobin have been measured in a wide range of solution conditions in the presence and absence of various allosteric effectors in order to determine how far hemoglobin can modulate its O2 affinity. The O2 affinity, cooperative behavior, and the Bohr effect of hemoglobin are modulated principally by tertiary structural changes, which are induced by its interactions with heterotropic allosteric effectors. In their absence, hemoglobin is a high affinity, moderately cooperative O2 carrier of limited functional flexibility, the behaviors of which are regulated by the homotropic, O2-linked T/R quaternary structural transition of the Monod-Wyman-Changeux/Perutz model. However, the interactions with allosteric effectors provide such “inert” hemoglobin unprecedented magnitudes of functional diversities not only of physiological relevance but also of extreme nature, by which hemoglobin can behave energetically beyond what can be explained by the Monod-Wyman-Changeux/Perutz model. Thus, the heterotropic effector-linked tertiary structural changes rather than the homotropic ligation-linked T/R quaternary structural transition are energetically more significant and primarily responsible for modulation of functions of hemoglobin. human adult hemoglobin Hb from which terminal His and Tyr are removed by caboxypeptidase A treatment Monod-Wyman-Changeux bezafibrate 2,3-diphosphoglycerate (or 2,3-biphosphoglycerate) inositol hexaphosphate Hemoglobin (Hb)1 has played a pivotal role in the understanding of the mechanisms of allosteric enzymes. Monod et al. (1Monod J. Wyman J. Changeaux P. J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Scopus (6057) Google Scholar) designated Hb an honorary enzyme, since it used the same molecule (O2) forsignaling as well as regulation. With the advent of detailed molecular structure at atomic levels, the question of enzyme activity became one of molecular mechanism. In the case of an allosteric enzyme, there is a need to assume at least two possible structures, customarily labeled T and R (1Monod J. Wyman J. Changeaux P. J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Scopus (6057) Google Scholar), and to regulate ligand affinity in each structure. The first question of signalingis straightforward, for it involves alternate packing of interfaces, for example. Monod’s original proposal (1Monod J. Wyman J. Changeaux P. J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Scopus (6057) Google Scholar) to assign deoxy- and oxy-Hbs to the T and R states acquired a structural foundation, since crystallographic studies (2Muirhead H. Perutz M.F. Nature. 1963; 199: 633-638Crossref PubMed Scopus (148) Google Scholar) revealed that the three-dimensional molecular structures of deoxy-Hbs and ligated Hbs. The hemoglobin molecule, a heterotetramer, consists of two α- and two β-subunits, each of which contains one O2-binding heme group. These four subunits are paired as two dimers, α1β1 and α2β2. The structural studies showed that deoxy-Hbs and ligated Hbs have two different modes of packing of the two dimers (the quaternary structures) with no major changes in the gross conformation of each of the subunits (the tertiary structures). Thus, Perutz (3Perutz M.F. Nature. 1970; 228: 726-739Crossref PubMed Scopus (2188) Google Scholar) assigned “deoxy” and “oxy” Hbs to T and R quaternary states, which exhibited low and high O2 affinity, respectively. The second question concerning regulation is the deeper one, and for Hb it has proved remarkably elusive. The essence of the question is to find a way in the low affinity T (deoxy) state to store free energy that is made available to bind ligands in the high affinity R (oxy) state. The MWC/Perutz stereochemical model (1Monod J. Wyman J. Changeaux P. J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Scopus (6057) Google Scholar, 3Perutz M.F. Nature. 1970; 228: 726-739Crossref PubMed Scopus (2188) Google Scholar), in which the O2 affinity of Hb is regulated primarily by the T/R quaternary transition, approximates well the behaviors of Hb under physiological conditions. Therefore, Perutz (3Perutz M.F. Nature. 1970; 228: 726-739Crossref PubMed Scopus (2188) Google Scholar) made the first proposal that salt bridges (formation/dissolution) are the key to regulation. Difficulty in establishing the centrality of the salt bridges led Perutz to propose that the affinity was lowered by storing energy as strain at the heme moiety, perhaps because of the spin radius of the high spin iron. However, spin energy and heme strains store insufficient energy to account for this cooperativity. Hopfield (4Hopfield J. J. Mol. Biol. 1973; 77: 208-222Crossref Scopus (118) Google Scholar) proposed a distributed strain model in which small strains throughout the protein stored the energy difference. Molecular modeling led Gelinet al. (5Gelin B.R. Lee A.W. Karplus M. J. Mol. Biol. 1983; 171: 489-559Crossref PubMed Scopus (137) Google Scholar) to propose that the movement of the proximal histidine pulling on the F-helix was a locus of strain energy storage. Related proposals based upon resonance Raman data were put forth by Nagai et al. (6Nagai K. Kitagawa T. Morimoto H. J. Mol. Biol. 1980; 136: 271-289Crossref PubMed Scopus (205) Google Scholar) and by Friedman et al. (7Friedman J. Scott T.W. Stepnoski R.A. Ikeda-Saito M. Yonetani T. J. Biol. Chem. 1985; 258: 10564-10572Abstract Full Text PDF Google Scholar). Nonetheless, no definitive experiment has successfully ascribed to any structural feature the amount of energy sufficient to account for the difference in the O2 affinity between T (deoxy) and R (oxy) structures of Hb. We have measured O2-binding curves of Hb in a wide range of solution conditions in the presence and absence of various heterotropic allosteric effectors. These measurements have been intended to probe the following questions: (i) how far Hb can modulate its O2affinity, (ii) how its cooperativity and its Bohr effect are modulated, and (iii) by what mechanism such modulation of the O2affinity is carried out. We have demonstrated that the O2affinity, the cooperativity, and the Bohr effect of Hb are modulated in hitherto unimaginable extents by the interactions of the allosteric effectors with Hb, especially in their interactions with oxy-Hb. This has led us to an inevitable conclusion that the substantial amount of the free energy of cooperativity/allostery must be stored in the binding of the various effectors or in the tertiary structural constraints, imposed by the interactions with the effectors. We show that the observed diverse behaviors of Hb are energetically beyond what can be explained by the MWC/Perutz T/R quaternary transition model (3Perutz M.F. Nature. 1970; 228: 726-739Crossref PubMed Scopus (2188) Google Scholar). Human blood samples were purchased from the local blood bank branch of the American Red Cross, from which Hb was purified according to the method of Drabkin (8Drabkin D. J. Biol. Chem. 1946; 164: 703-723Abstract Full Text PDF PubMed Google Scholar) and stripped of organic phosphates by the method of Berman et al. (9Berman M. Benesch H. Benesch R.E. Arch. Biochem. Biophys. 1971; 145: 236-239Crossref PubMed Scopus (118) Google Scholar). Des-Hisβ146,Tyrβ145-Hb, or des-His-Tyr-Hb, was prepared by the digestion of Hb with carboxypeptidase A (Sigma) at a ratio of enzyme/Hb (w/w) of 0.1 for 4 h at 30 °C in 0.05 m Tris-HCl buffer, pH 8.0 (10Antonini E. Wyman J. Zito R. Rossi-Fanelli A. Caputo A. J. Biol. Chem. 1961; 236: PC60-PC63Abstract Full Text PDF PubMed Google Scholar). The digestion was terminated by the addition of 1 mmhydrocinnamic acid. The Hb preparation thus treated was separated from released amino acids by a molecular sieve through a column of Sephadex G-25 equilibrated with 10 mm bis-Tris propane buffer, pH 7.4. Oxygen equilibria were measured by an improved version of Imai’s automatic polarography-spectrophotometry method (11Imai K. Morimoto H. Kotani M. Watari H. Hirata W. Kuroda M. Biochim. Biophys. Acta. 1970; 200: 189-196Crossref PubMed Scopus (299) Google Scholar, 12Imai K. Yonetani T. Biochim. Biophys. Acta. 1977; 490: 164-170Crossref PubMed Scopus (61) Google Scholar, 13Imai K. Allosteric Effects in Hemoglobin. Cambridge University Press, Cambridge, UK1982Google Scholar, 14Imai K. Yonetani T. J. Biol. Chem. 1975; 250: 2227-2231Abstract Full Text PDF PubMed Google Scholar) with the following modification. Absorbance was monitored using a computer-controlled Olis-Cary 118 spectrophotometer (Olis, Bogart, Georgia). Oxygen concentrations were monitored with a low zero current, rapid response electrode (O2 sensors; Gladwyne, PA), using a custom-made amplifier (Biomedical Instrumentation Shop, University of Pennsylvania Medical Center). The signal was then digitized using a 12-bit A/D converter. Absorption spectra of oxy-Hb and deoxy-Hb in the visible region were independent of pH and the heterotropic effectors used. Therefore, absorbance changes at 560 nm were proportional only to the degrees of O2 saturation of Hb. The degrees of fractional O2 saturation (the Y values) were computed from the changes in absorbance at 560 nm by getting the absorbance values at the fully deoxy and oxy states by extrapolation with precautions previously described (13Imai K. Allosteric Effects in Hemoglobin. Cambridge University Press, Cambridge, UK1982Google Scholar, 14Imai K. Yonetani T. J. Biol. Chem. 1975; 250: 2227-2231Abstract Full Text PDF PubMed Google Scholar). The O2 equilibrium data were expressed as Hill plots of log(Y/(1 − Y))versus log pO2. The O2 equilibrium measurements were carried out in 0.1 m HEPES buffer (pH 6.6–9.0) at 15 °C. The concentrations of reactants used were 60 μm (heme) Hb, 0.1 m Cl−, 10 mm BZF, 2 mm DPG, and 2 mm IHP. We had previously shown that the effects of the tetramer-dimer dissociation of Hb on O2 equilibrium parameters were mimimally affected at [Hb] ≥ 60 μm (12Imai K. Yonetani T. Biochim. Biophys. Acta. 1977; 490: 164-170Crossref PubMed Scopus (61) Google Scholar). Oxygen binding curves at each of 168 different solution conditions were measured in triplicates in a cycle of “deoxygenation followed by reoxygenation” processes in order to assure the viability of the Hb preparation and the measuring conditions. The formation of methemoglobin after each measurement was less than 3%. Oxygen binding data were analyzed by a nonlinear least square curve-fitting analysis according to the Adair scheme (15Adair G.S. J. Biol. Chem. 1925; 63: 529-545Abstract Full Text PDF Google Scholar) using Equation 1, Y=K1p+3K1K2p2+3K1K2K3p3+K1K2K3K4p41+4K1p+6K1K2p2+4K1K2K3p3+K1K2K3K4p4Equation 1 where Y and p are the degree of O2 saturation and the partial pressure of O2, respectively, and K1, K2,K3, and K4 are the intrinsic O2 equilibrium association constants at oxygenation steps 1, 2, 3, and 4, respectively. Processed oxygenation data were arranged in the form (log p, log[Y/(1 − Y)]) and fitted with the custom-made nonlinear curve fitting procedure in Origin version 6.1 (Microcal, Northampton, MA), according to Equation 1. Initial values for K1 and K4 could be readily obtained graphically from the lower and upper asymptotes of the Hill plots, respectively, whereas initial estimates forK2 and K3 were arbitrarily made from experimental data at ∼30 and ∼60% saturation, respectively. Sets of fitted values forK1, K2,K3, and K4 were obtained after the convergence of successive iterations was achieved. The parameter ΔH+average is the average number of Bohr protons released (expressed as negative value) per binding site, which are numerically calculated from averaging the number of Bohr protons released at each step of four Adair O2 binding equilibria (14Imai K. Yonetani T. J. Biol. Chem. 1975; 250: 2227-2231Abstract Full Text PDF PubMed Google Scholar, 16Tsuneshige A. Park S.I. Yonetani T. Biophys. Chem. 2002; 98: 49-63Crossref PubMed Scopus (58) Google Scholar). A nonlinear least-squares regression curve-fitting analyses of O2equilibrium data were also performed according to the MWC allosteric model using Equation 2 (1Monod J. Wyman J. Changeaux P. J. Mol. Biol. 1965; 12: 88-118Crossref PubMed Scopus (6057) Google Scholar), Y=L0KTp(1+KTp)3+KRP(1+KRP)3L0(1+KTp)4+(1+KRp)4Equation 2 where Y represents the degree of saturation with O2, p is the partial pressure of O2,KT and KR are O2 association equilibrium constants of T (deoxy) and R (oxy) states, respectively, and L0 = [T0]/[R0] and L4 = [T4]/[R4] =L0(KT/KR)4, allosteric equilibrium constants, where [T0], [R0], [T4], and [R4] stand for equilibrium molar concentrations of T (deoxy) and R (deoxy) conformers in the deoxy state and those of T (oxy) and R (oxy) conformers in the fully oxy state, respectively. Processed oxygenation data were arranged in the form (log p, log[Y/(1 − Y)]) and fitted with the custom-made nonlinear curve fitting procedure in Origin version 6.1 (Microcal), according to Equation 2. Initial estimates forKT, and KR could be readily obtained graphically from Hill plots or fitted data sets according to the Adair scheme. Namely, KT andKR can be estimated from the lower and upper asymptotes on the Hill plots, which can be approximated toK1 and K4, respectively. The L0 values were estimated with an approximation of L0 = (K4× P50)4 (16Tsuneshige A. Park S.I. Yonetani T. Biophys. Chem. 2002; 98: 49-63Crossref PubMed Scopus (58) Google Scholar). Unique sets of fitted values for KT, KR, andL0 were thus obtained after the convergence of successive iterations was achieved. Although Marden et al.(17Marden M.C. Bohn B. Kister J. Poyart C. Biophys. J. 1990; 57: 397-403Abstract Full Text PDF PubMed Scopus (52) Google Scholar) analyzed their O2 binding data and concluded thatKR cannot be deduced from their O2binding equilibrium curves, our successive iterations using 10-fold increased or decreased initial values of KT,KR, or L0 were found not to alter the values of KT,KR, and L0 at the convergence. This was perhaps due to our more extensive collection of accurate O2 binding data at higher saturation levels using the substantially improved instrumentation. The free energy of cooperativity (ΔG0) was calculated from ΔG0 = −2.3RTlog(KR/KT). The NMR experiments were made with a Bruker ARX-500 NMR spectrometer at 15 and 29 °C, using 2 mm (heme) Hb, 10 mm BZF, 10 mm IHP, 0.1 m Cl−, and 0.1 m HEPES buffer, pH 7.0, in 90% H2O, 10% D2O. The molar ratio of [effector]/[Hb] of 5 used for NMR is less than those used in the oxygenation experiments. However, actual degrees of saturation of Hb with effectors in NMR experiments were substantially more than those of the oxygenation experiments, since the saturation depends on the concentrations of the reactants rather than the molar ratio as long as [effector] > [Hb] or free effectors are available. The water signal was suppressed by using a jump-and-return pulse sequence (18Plateau P. Gueáron M. J. Am. Chem. Soc. 1982; 104: 7310-7311Crossref Scopus (1137) Google Scholar). Proton chemical shifts were referred to internal sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4. In addition to exchangeable proton NMR spectroscopy in the hydrogen-bonded region (19Shulman R.G. Hopfield J.J. Ogawa S. Annu. Rev. Biophys. 1975; 8: 325-420Google Scholar, 20Dalvit C. Ho C. Biochemistry. 1985; 24: 3398-3407Crossref PubMed Scopus (58) Google Scholar, 21Russu I.M., Ho, N.T. Ho C. Biochim. Biophys. Acta. 1987; 914: 40-48Crossref PubMed Scopus (47) Google Scholar), the quaternary state of Hb in solution was probed by the following techniques: (i) UV fine structure difference spectrophotometry (22Imai, K., and Yonetani, T. (197) Biochem. Biophys. Res. Commun.50, 1055–1060Google Scholar, 23Imai K. Biochemistry. 1973; 12: 128-134Crossref PubMed Scopus (19) Google Scholar) using a Hewlett-Packard 8452A diode array spectrophotometer, (ii) UV-CD spectrophotometry (24Simon S.R. Cantor C.R. Proc. Natl. Acad. Sci. U. S. A. 1969; 63: 205-212Crossref PubMed Scopus (46) Google Scholar) using an AVIV 62DS CD spectrophotometer, and (iii) spectrophotometric assay of the reactivity of the (Cysβ93-SH groups (16Tsuneshige A. Park S.I. Yonetani T. Biophys. Chem. 2002; 98: 49-63Crossref PubMed Scopus (58) Google Scholar,25Grassetti D.R. Murray J.F. Jr. Arch. Biochem. Biophys. 1967; 119: 41-49Crossref PubMed Scopus (868) Google Scholar, 26Imai K. Hamilton H.B. Hiyaji T. Shibata S. Biochemistry. 1972; 11: 114-121Crossref PubMed Scopus (20) Google Scholar, 27Yonetani T. Tsuneshige A. Zhou Y. Chen X. J. Biol. Chem. 1998; 273: 20323-20333Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 28Imaizumi K. Imai K. Tyuma I. J. Biochem. (Tokyo). 1979; 86: 1829-1840Crossref PubMed Scopus (42) Google Scholar). In order to measure the reversible O2 binding curves of Hb in the widest possible range of well controlled conditions, we made extensive examinations of experimental conditions that are optimally suited. The HEPES buffer system was chosen among many buffers commonly used in Hb research (phosphate, Tris-Cl−, bis-Tris-Cl−, bis-Tris propane, HEPES, etc.) for the following reasons. The buffering capability of HEPES covers a reasonably wide range of physiological pH (pH 6.6–9.0). The HEPES buffers keep all of the allosteric effectors used soluble in the entire pH range at 15 °C. The HEPES buffers neither interact with the allosteric effectors used nor alter Hb irreversibly and/or nonspecifically in the entire range of pH employed. The HEPES buffers exhibit no detectable allosteric effect on Hb at the concentration employed. Although the majority of previous Hb works had been carried out at higher temperatures (20, 25, or 37 °C), we had chosen the measuring temperature at 15 °C, which was an optimal compromise of competing requirements to obtain quantitatively full O2 binding curves under our widely ranging experimental conditions. Since we dealt with Hb at low affinity (P50 of up to 150 torr), nearly full oxygenation was feasible only at lower temperatures (≤15 °C) even at an atmospheric pressure of 100% O2 (the maximal O2 pressure used in our experiments of pO2 = 760 torr minus saturated aqueous vapor pressure). On the other hand, deoxygenation was more readily accomplished at higher temperatures, especially for high affinity states of Hb (P50 = 0.1–0.5 torr). Furthermore, the formation of met Hb during measurements was greatly reduced at this temperature. The Cl− concentration was fixed at 0.1 m (the physiological concentration). The concentrations of other allosteric effectors were set at 2 mm DPG, 2 mm IHP, and 10 mm BZF. These concentrations were chosen to maintain the effectors soluble in the entire pH range used at 15 °C. Thus, [effector] was always in large excess over [Hb], except for [H+], which was varied from 10−9 to 10−6.6m. Figs. 1 and 2illustrate Hill plots of the O2 binding data in the absence and presence of 0.1 m Cl−, respectively. It is noted that the Hill plots in the absence of heterotropic allosteric effectors above pH 7.8 are fully superimposable with a fixed cooperativity of nmax = 2.5 (Fig.1A). However, as pH is decreased below pH 7.8, the Hill plots shift toward the right with slight downward shifts of the lower and upper asymptotes without changes in the cooperativity. In the presence of other heterotropic allosteric effectors, these pH-dependent shifts of the Hill plots become more and more pronounced in the order of apparent allosteric potencies of H+ < Cl− < DPG < BZF < IHP < BZF + DPG < BZF + IHP under the prescribed concentrations of the effectors (Figs. 1 and2). The downward shift of the lower asymptotes approach and eventually go beyond an apparent minimal value of P50 = 100 torr (the lower dotted line) in the presence of the maximal allosteric effect (with BZF + IHP at pH 6.6) (Fig. 1F). The downward shifts of the upper asymptotes are accompanied by gradual decrease of the apparent cooperativity (the slopes of the middle portion of Hill plots). When the upper asymptotes reach the minimal value of P50 = 100 torr, the apparent cooperativity is reduced to nmax ≈ 1 at the maximal allosteric constraint (Figs. 1F and2F).Figure 2Hill plots of O2 binding equilibria of Hb in the presence of 0.1 mCl−at different pH values with and without heterotropic effectors.Symbols andpanels are as in Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Thus, the O2 affinity of Hb is modulated as much as >1,000-fold from P50 ≈ 0.1 torr (the upper asymptotes in Fig. 1A) to P50≈ 160 torr (the lower asymptotes in Fig. 1F) by the influence of heterotropic allosteric effectors. Comparison of the Hill plots in Figs. 1 and 2 provides the following conclusions. (i) The allosteric effect of H+ is additive or synergetic with those of all other heterotropic effectors. (ii) The allosteric effect of Cl− is competitive or antagonistic against those of other heterotropic effectors, as previously observed by others (13Imai K. Allosteric Effects in Hemoglobin. Cambridge University Press, Cambridge, UK1982Google Scholar, 28Imaizumi K. Imai K. Tyuma I. J. Biochem. (Tokyo). 1979; 86: 1829-1840Crossref PubMed Scopus (42) Google Scholar) (Fig. 1versus Fig. 2). (iii) The effect of BZF is additive or synergetic with organic phosphates (DPG or IHP) (Fig. 1, E and F, and Fig. 2, E andF), as previously noted (17Marden M.C. Bohn B. Kister J. Poyart C. Biophys. J. 1990; 57: 397-403Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 29Perutz M.F. Poyart C. Lancet. 1983; ii: 881-882Abstract Scopus (95) Google Scholar, 30Marden M.C. Kister J. Bohn B. Poyart C. Biochemistry. 1988; 27: 1659-1664Crossref PubMed Scopus (81) Google Scholar, 31Coletta M. Angeletti M. Ascenzi P. Bertollini A. Longa S.D., De Sanctis G. Priori A.M. Santucci R. Amiconi G. J. Biol. Chem. 1999; 274: 6865-6874Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). (iv) The O2 affinity of unmodified Hb has an apparent lowest limit at P50 ≈ 160 torr under the present solution conditions. (v) The Bohr effect of Hb (or the pH dependence ofP50) is increasingly enhanced by the heterotropic effectors in the order of their apparent allosteric potencies mentioned above. The O2 binding data shown in Figs. 1 and 2 have been analyzed by Equation 2 of the MWC model. Fig. 3lists the MWC parameters and related oxygenation parameters (ΔG0, P50, andnmax) primarily from those in the presence of Cl− (Fig. 1A and Fig. 2, A–F). The MWC and Adair parameters obtained are comparable with those reported previously in the literature (13Imai K. Allosteric Effects in Hemoglobin. Cambridge University Press, Cambridge, UK1982Google Scholar). The Hill plots of Figs. 1 and 2 are fully and quantitatively simulated by these MWC parameters (KT, KR, andL0) as well as by the Adair constants (K1, K2,K3, and K4). For example, Fig. 4 illustrates simulation curves of Hill plots according to MWC and Adair models. TableI lists the selected sets of the MWC parameters and Adair constants in the presence of Cl−, BZF, and IHP, which were used for Fig. 4. The correlation of the MWC parameters is also expressed by the “global allostery” plots, namely the log K versus log L plots (32Imai K. Tsuneshige A. Yonetani T. Biophys. Chem. 2002; 98: 79-91Crossref PubMed Scopus (20) Google Scholar, 33Imai K. J. Mol. Biol. 1983; 167: 741-749Crossref PubMed Scopus (31) Google Scholar) in Figs. 5 and6. Such plots give explicitly the correlation among the MWC parameters under different effector conditions. The concentrations of allosteric effectors used are below saturation. However, in the MWC model, it is not the necessary condition that every state must be saturated with the effectors present in the solution. The important condition is that the concentration of free effectors must be “virtually constant” during the oxygenation process. This condition is well met in this study, since the [effector]/[Hb] ratio is sufficiently larger than unity. It should be pointed out that all of the values of the MWC parameters obtained at higher concentrations of effectors or with stronger effectors lie on the closed distorted circular plot of Fig. 6, although the values are found at higher pH than the corresponding values shown in Fig. 6. Thus, we consider the global allostery plot of Fig. 6 to be universally applicable to normal Hb under different concentrations of effectors and/or different effectors.Figure 4Simulation of Hill plots of O2-binding equilibria of Hb using MWC and Adair parameters of Table I.Symbols indicate experimental points (only 50% of the 90 data points in each experiment are shown), which were obtained in 0.1 m HEPES buffer, pH 6.6–9.0, in the presence of Cl−, BZF, and IHP at 15 °C (Fig.2F). Under the conditions, large (∼500-fold) pH-dependent modulations of KR were observed. Solid and broken curvesrepresent simulated Hill plots based upon numerical values of the MWC and Adair models, respectively (cf. Table I).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IComparison of MWC and Adair parameters of hemoglobin in O2-binding equilibriapHKTKRL0K1K2K3K4ΔH+average9.05.5E − 29.0E + 05.2E + 54.77E − 24.97E − 25.56E − 19.44E + 0−0.38.63.3E − 25.3E + 02.6E + 53.16E − 25.15E − 23.56E − 15.37E + 0−0.68.21.8E − 23.4E + 07.9E + 51.66E − 23.39E − 27.77E − 23.71E + 0−0.97.81.2E − 21.2E + 03.8E + 51.15E − 21.85E − 22.46E − 29.52E − 1−1.17.41.0E − 21.3E − 11.6E + 39.20E − 31.30E − 21.64E − 28.71E − 2−0.77.07.0E − 33.0E − 23.3E + 17.20E − 31.05E − 21.15E − 22.33E − 2−0.36.66.0E − 32.0E − 21.5E + 16.30E − 39.10E − 31.15E − 21.79E − 2−0.1Oxygen binding equilibria were measured in 0.1 m HEPES buffer, pH 6.6–9.0, in the presence of 0.1 m Cl−, 10 mm BZF, and 2 mm IHP at 15 °C. Open table in a new tab Figure 5The global allostery plots (log K versus log L plots) of MWC parameters of O2 binding equilibria of Hb. The numerical mark of each data point indicates the measuring pH (1, 2,3, 4, 5, 6, and7 represent pH 6.6, 7.0, 7.4, 7.8, 8.2, 8.6, and 9.0, respectively). A, with (▪) and without Cl−(◯); B, with Cl− + DPG (▪) and Cl− + BZF (◯); C, with Cl− + IHP (◯) and Cl− + IHP + BZF (▪); D, with IHP + BZF.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Summary of the global allostery plots (the log K versus log L plots) of MWC parameters of Hb (circles) and des-His-Tyr-Hb (squares) at pH 6.6–9.0. The effectors used arecolor-coded. The data points shift downward continuously as pH is decreased from pH 9.0 to 6.6 (see examples in Fig. 5).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Oxygen binding equilibria were measured in 0.1 m HEPES buffer, pH 6.6–9.0, in the presence of 0.1 m Cl−, 10 mm BZF, and 2 mm IHP at 15 °C. Exchangeable proton NMR spectra in the hydrogen-bonded region of deoxy-Hb and ligated Hb were measured at pH 7.0 with and without allosteric effectors at 15 °C (Fig.7) and 29 °C. This was to determine whether the effector-induced shifts in the allosteric equilibrium toward T (R0 → T0 and/or R4 → T4, respectively) would alter the apparent T/R quaternary states of deoxy- and oxy-Hb. The pattern of the NMR spectra representing the quaternary structure-specific hydrogen bonds (19Shulman R.G. Hopfield J.J. Ogawa S. Annu. Rev. Biophys. 1975; 8: 325-420Google Scholar, 20Dalvit C. Ho C. Biochemistry. 1985; 24: 3398-3407Crossref PubMed Scopus (58) Google Scholar, 21Russu I.M., Ho, N.T. Ho C. Biochim. Biophys. Acta. 1987; 914: 40-48Crossref PubMed Scopus (47) Google Scholar) is not altered in the presence of various allosteric effectors. The quaternary structures were also probed by other methods, which are considered to indicate the quaternary structures of Hb in solution. Ultraviolet absorption fine structures showed larger troughs at 294 nm on the first derivative display (ΔA294/Δλ) in R (ligated) states than in T (deoxy) states. They were not significantly affected by the addition of allosteric effectors (TableII). Cir" @default.
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• W1969361812 date "2002-09-01" @default.
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• W1969361812 title "Global Allostery Model of Hemoglobin" @default.
• W1969361812 cites W1506154845 @default.
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• W1969361812 cites W1603979102 @default.
• W1969361812 cites W1622780310 @default.
• W1969361812 cites W1641120577 @default.
• W1969361812 cites W1964804671 @default.
• W1969361812 cites W1968079241 @default.
• W1969361812 cites W1968360065 @default.
• W1969361812 cites W1972681415 @default.
• W1969361812 cites W1972919297 @default.
• W1969361812 cites W1973175248 @default.
• W1969361812 cites W1973243449 @default.
• W1969361812 cites W1974131004 @default.
• W1969361812 cites W1975658156 @default.
• W1969361812 cites W1981622771 @default.
• W1969361812 cites W1983979304 @default.
• W1969361812 cites W1994067305 @default.
• W1969361812 cites W1994088755 @default.
• W1969361812 cites W1995560641 @default.
• W1969361812 cites W1996532134 @default.
• W1969361812 cites W1998093640 @default.
• W1969361812 cites W1998876039 @default.
• W1969361812 cites W1999451343 @default.
• W1969361812 cites W2005392755 @default.
• W1969361812 cites W2006815507 @default.
• W1969361812 cites W2009505756 @default.
• W1969361812 cites W2011836815 @default.
• W1969361812 cites W2013334845 @default.
• W1969361812 cites W2014790347 @default.
• W1969361812 cites W2024191400 @default.
• W1969361812 cites W2026237439 @default.
• W1969361812 cites W2026911129 @default.
• W1969361812 cites W2031079498 @default.
• W1969361812 cites W2031081445 @default.
• W1969361812 cites W2032372720 @default.
• W1969361812 cites W2036474910 @default.
• W1969361812 cites W2040881232 @default.
• W1969361812 cites W2053982041 @default.
• W1969361812 cites W2059243326 @default.
• W1969361812 cites W2062495744 @default.
• W1969361812 cites W2068758907 @default.
• W1969361812 cites W2084389878 @default.
• W1969361812 cites W2085496821 @default.
• W1969361812 cites W2090243522 @default.
• W1969361812 cites W2113974771 @default.
• W1969361812 cites W2127598700 @default.
• W1969361812 cites W2167653651 @default.
• W1969361812 cites W230984257 @default.
• W1969361812 cites W3005306837 @default.
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