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Protein Science (2002), 11:1845-1849.
Copyright © 2002 The Protein Society

FOR THE RECORD

Correlation of protein functional properties in the crystal and in solution: The case study of T-state hemoglobin

Robert W. Noble1, Laura D. Kwiatkowski1, Hilda L. Hui1, Stefano Bruno2, Stefano Bettati2,3,4 and Andrea Mozzarelli2,4

1 Department of Medicine, University at Buffalo, Veterans Administration Medical Center, Buffalo, New York 14215, USA
2 Department of Biochemistry and Molecular Biology, University of Parma, 43100 Parma, Italy
3 Department of Public Health, University of Parma, 43100 Parma, Italy
4 Italian National Institute for the Physics of Matter, University of Parma, 43100 Parma, Italy

Reprint requests to: Robert W. Noble, Department of Medicine, University at Buffalo (SUNY), Buffalo VA Medical Center, 3495 Bailey Ave., Buffalo, New York 14215, USA; e-mail: rnoble{at}acsu.buffalo.edu; fax: (716) 862-6526.

(RECEIVED February 27, 2002; FINAL REVISION April 9, 2002; ACCEPTED April 25, 2002)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0205702.


   Abstract
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 Abstract
Introduction
 Results and Discussion
 Materials and methods
 References
 
The relevance of three-dimensional structures of proteins, determined by X-ray crystallography, is an important issue that is becoming even more critical in light of the Structural Genomics Initiative. As a case study, a detailed comparison of functional properties of the T quaternary states of genetically or chemically modified human hemoglobins (Hbs) in solution and in the crystal was performed. Oxygen affinities of Hbs in crystals correlate with the rate constants of their initial combination with carbon monoxide (CO) in solution, indicating that changes in ligand affinity caused by the modifications are similarly observed in both physical states.

Keywords: Hemoglobin; T quaternary structure; ligand affinity; mutational effects; properties in solution; properties in crystals

Abbreviations: PEG, polyethylene glycol • IHP, inositol hexaphosphate • HbA, human adult hemoglobin • Hb, hemoglobin • desArg, human hemoglobin from which the C-terminal arginines of the {alpha} subunits have been enzymatically removed • desHis, human hemoglobin from which the C-terminal histidines of the ß subunits have been removed • deoxyHb, deoxygenated or unliganded hemoglobin


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Materials and methods
 References
 
Most of the information currently available about the three-dimensional structures of proteins results from crystallographic analysis. The question of the relationship between the structure and function of proteins in crystals and in solution has been and remains a matter of interest (Parkhurst and Gibson 1967; Mozzarelli and Rossi 1996). The issue of function within the crystalline state has been addressed using several methodological approaches: kinetic studies on microcrystalline protein suspensions, single crystal spectroscopy and microspectroscopy, fluorometry, infrared, Raman, and resonance Raman spectroscopy (Mozzarelli and Bettati 2001). A particularly powerful and flexible technique is polarized absorption microspectrophotometry with which kinetic, thermodynamic, and structural parameters can be measured on single crystals of the same size as those used for X-ray crystallography. In recent years, this technique has been used to characterize several redox and catalytic proteins (Mozzarelli and Rossi 1996; Mozzarelli and Bettati 2001) and, in particular, the equilibria of oxygen binding to hemoglobins (Hbs). Mozzarelli et al. (1996) have shown that crystals of the dimeric Hb of Scapharca inaequivalvis bind oxygen with full cooperativity. This shows that complex functional properties can be preserved in the crystalline state provided that the structural transitions involved are not restricted by lattice interactions. In human Hb, cooperativity is associated with substantial changes in quaternary structure, involving the transition from the deoxygenated T quaternary state, with its low ligand affinity, to the fully liganded R quaternary state with its high affinity. The equilibrium of oxygen binding to T-state human adult hemoglobin (HbA) crystals has been examined in considerable detail (Mozzarelli et al. 1991; Rivetti et al. 1993a; Eaton et al. 1999). This binding equilibrium is characterized by a Hill coefficient very near unity and a very low affinity, which is unaffected by strong allosteric effectors such as inositol hexaphosphate (IHP) or by changes in pH (Mozzarelli et al. 1997). The properties of the T quaternary structure in solution, as judged by the equilibria and kinetics of the binding of the first ligand to the deoxygenated tetramer, are not constant but depend on solution conditions. IHP acts to substantially reduce the ligand affinity of the T state. The affinity of T-state crystals of HbA approximates that of the equilibrium constant, K1, for the reaction of the first oxygen molecule with HbA in solution in the presence of IHP (Poyart et al. 1978; Imai 1982). The affinity of the crystals also approximates that reported by Bruno et al. (2001) for the low affinity T-state Hb isolated by encapsulation of deoxyHbA in wet silica gels in the presence of IHP and bezafibrate. It has been proposed (Mozzarelli et al. 1997) that crystallization selects for the low affinity conformers of the T quaternary state, the same conformers to which IHP binds preferentially in solution. If this is so, then it would seem that mutations and other chemical modifications should have the same effects on the properties of the crystalline T state as they have on the T state in solution in the presence of IHP.

Changes in oxygen affinity of Hb are closely paralleled by changes in the affinity for carbon monoxide (CO) in solution (Tan et al. 1973; Ackers 1998). Thus, CO is considered a very good analog of oxygen. Changes in CO affinity are reflected by changes in the second order rate constant for the combination of CO. The low affinity, deoxygenated T quaternary structure of HbA binds CO some 30-fold more slowly than the high affinity R quaternary state or {alpha}ß dimers (Gibson 1959; Edelstein et al. 1970). The tertiary Bohr effect in K1, that is, the influence of proton on ligand binding to T-state Hb in solution (Imai and Yonetani 1975), is accompanied by a clear pH dependence in the rate constant for CO combination (Pennelly and Noble 1978; McDonald et al. 1979). In addition, Schreiber and Parkhurst (1984) have pointed out that the correlation between CO affinity and CO combination rate extends to a wide variety of diverse, noncooperative heme proteins. Therefore, the rate of CO combination with deoxygenated HbA, deoxyHbA, offers a convenient parameter with which to approximate changes in the ligand affinity of the deoxyHb tetramer in solution.

Since the first measurements of the oxygen equilibria of crystals of HbA (Mozzarelli et al. 1991), similar measurements have been performed on a series of chemically or genetically modified Hb molecules. With concomitant measurements of the kinetics of CO combination with the deoxygenated derivatives of these Hb molecules in solution, there are now sufficient data to permit the examination of the extent to which these two parameters are correlated.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Materials and methods
 References
 
The oxygen affinities of HbA crystals in the absence and presence of strong allosteric effectors and crystals of several HbA mutants, obtained by chemical or genetic modifications, have recently been determined (Rivetti el al. 1993a ,b ;Kavanaugh et al. 1995, 2001; Bettati et al. 1997; Mozzarelli et al. 1997; Noble et al. 2001). We have chosen to test the correlation between the oxygen affinities of Hb crystals and the kinetic properties of T states in solution in the presence of IHP. This has the additional benefit that IHP greatly stabilizes the T-state tetramer as a result of its preferential binding to this structure. The deoxygenated tetramers of some of the Hb variants we have examined are destabilized to the extent that under the conditions of the kinetic measurements a significant fraction of the deoxyHb molecules are dissociated into {alpha}ß dimers. The presence of IHP effectively eliminates this dissociation, permitting the examination of the kinetic properties of the Hb tetramer without the complicating presence of the rapidly reacting dimers (Noble et al. 2001).

In Figure 1Go the logarithm of the partial pressure of oxygen required for half saturation of Hb crystals grown from polyethylene glycol (PEG) is plotted as a function of the logarithm of the initial second order rate constant, l`init, for CO combination with deoxyHbs in solution. The logarithmic plot is chosen because of the proportionality of log p50 to the {Delta}G° of the equilibrium process and of log l`init to the activation energy of the combination reaction. The line represents the linear least squares fit to the data points. The oxygen affinities of the crystals examined vary over a range of almost 60-fold. The rate constants for CO combination vary by as much as a factor of 20. Over this range of values, the two logarithmic functions show a linear correlation. The slope of the line indicates that on average a 10-fold increase in the oxygen affinity of the crystalline T state is associated with an approximately five-fold increase in the rate of CO combination with the deoxygenated T state in solution in the presence of IHP.



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  		Fig. 1. Correlation between the oxygen affinities of T state crystals and the initial rates of carbon monoxide (CO) combination with human adult hemoglobin (HbA) and a series of chemical or genetic variants of this protein. The logarithm of l`init, the rate constant for the initial reaction of CO with the deoxygenated hemoglobins (Hbs), is plotted as a function of the logarithm of p50(O2), the partial pressure of oxygen required for half saturation of the T-state crystals.

 
The correlation is good over the large range but is far from perfect with respect to small variations in the two parameters. A perfect fit would require that changes in {Delta}G° always be divided in the same proportion between the activation energies of the on and off kinetic reactions. This may not be precisely true. Deviations from the log–log relationship could also result in part from differences in the functional properties of the {alpha} and ß subunits and the difference in the averaging of the subunit properties that results from the two parameters being examined. The p50 of an equal mixture of two dissimilar binding sites is equal to the square root of the product of the p50 values for the two sites. On the other hand, the CO combination rate yields an arithmetic average of the rate constants of the two types of subunits. Another possibility is indicated by the realization that the two processes being compared, ligand binding to the crystalline T state and the initial attachment of a ligand to the deoxygenated Hb molecule in solution, have an important intrinsic difference. The absence of a Bohr effect in the binding of oxygen to the crystalline T state of Hb implies, and crystallographic examination confirms (Paoli et al. 1996; Luisi et al. 1990), that in the crystal, ligand binding is not associated with disruption of the salt bridges, which are associated with ligand-linked proton release. In contrast, K1 in solution has a significant Bohr effect (Imai and Yonetani 1975; Russo et al. 2001), and this is reflected in a pH dependence in the rate constant for CO combination with deoxyHb (Pennelly and Noble 1978; McDonald et al. 1979). This indicates that in solution, binding a ligand to the deoxyHb molecule involves pH- and ligand-dependent ionizations that do not occur in the crystal. To the extent that a mutation alters these ionizations, it can be expected to have a differential effect on the properties of the T state in the crystal and in solution. It is perhaps notable that the two variants, which deviate most from the fitted line in Figure 1Go, are desArg and desHis. Both the C terminal arginine of the {alpha} subunit and the C terminal histidine of the ß subunit have been implicated in the Bohr effect (Kilmartin and Wootton 1970; Bonaventura et al. 1974; Kilmartin et al. 1975), either as a direct source of Bohr protons or through indirect global electrostatic effects (Matthew et al. 1979; Matthew et al. 1982; Sun et al. 1997). Despite imperfections in the correlation between the parameters examined, it is clear from these results that the properties of the T quaternary structure in crystals grown from PEG solutions reflect those of the deoxygenated T state in solution in the presence of the strong allosteric effector IHP. These results are consistent with the crystal lattice selecting for a low affinity subset of the T state conformations, which exist in solution. In solution this low affinity subset must preferentially bind IHP and be selectively populated in its presence.

The fidelity of the reproduction of mutational effects, in going from solution to crystal, is potent evidence of functional identity in these two milieus. The maximum affinity change reported here corresponds to a change in the standard free energy of oxygen binding of <2.5 kcal/mole of heme. The changes in the activation energy of the initial CO combination reaction are even smaller. It is hard to imagine such a precise reproduction of mutational effects without great structural similarity in these two environments.


   Materials and methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
References
 
The genetic variants of HbA that are examined here were prepared as previously described (Hernan et al. 1992; Hui et al. 1999; Noble et al. 2001). DesHis is HbA from which the C terminal histidine residues of the ß subunits have been removed, and desArg is HbA from which the C terminal arginine residues of the {alpha} subunits have been removed. Both are the result of enzymatic hydrolysis (Kavanaugh et al. 1995; Bettati et al. 1997).

Measurements of equilibria of oxygen binding to crystals of deoxyHb were performed at 15°C as described by Rivetti et al. (1993a). The rate of the combination of CO with deoxyHb was measured at 20°C by stopped flow procedures in 100 mM HCl-bisTris buffer, 100 µM IHP at pH 7, as described by Doyle et al. (1992). The Hb concentration after mixing was 2 µM in heme equivalents and that of CO was 20 µM.

Most of the data being examined in this paper are taken from a series of articles that appeared in the past decade, beginning with the reports of the oxygen-binding properties of crystals of deoxyHbA grown from PEG solutions (Mozzarelli et al. 1991; Rivetti et al. 1993a). Oxygen equilibrium data have been reported for the crystals of desArg (Kavanaugh et al. 1995) and desHis (Bettati et al. 1997), for the ({alpha})2(ßY35A)2 and ({alpha})2(ßY35F)2 variants (Kavanaugh et al. 2001) and for ({alpha})2(ßW37E)2, ({alpha})2(ßN102A)2, and ({alpha})2(ßN108G)2 (Noble et al. 2001). Studies on the equilibria of oxygen binding to crystals of Hb Rothschild, ({alpha})2(ßW37R)2, have also been reported (Rivetti et al. 1993b). Unlike the other Hb variants described here, the oxygen affinity of crystals of Hb Rothschild changes in response to solution conditions. Specifically, this variant has a chloride ion-binding site at the mutant arginine residue (Kavanaugh et al. 1992), and the binding of chloride ions is negatively linked to the binding of ligand both at the {alpha} and at the ß hemes. Included in the comparison here are the measurements reported for Hb Rothschild crystals in chloride-free dilute phosphate buffer and in the standard chloride containing bisTris buffer. However, measurements of oxygen equilibria of crystals of Hb Rothschild were performed at 21°C, whereas all other measurements of O2-binding equilibria were performed at 15°C. Taking the decrease in the oxygen affinity of HbA with a 10° increase in temperature to be a factor of two (Imai 1982), this 6° difference was corrected by subtracting 0.18 from log p50 measured at 21°C.

Data reported here for crystals of ({alpha})2(ßN108L)2, ({alpha}Y42A)2(ß)2, and ({alpha})2(ßY145A)2 have not been reported previously. Crystals of the latter variant are uniquely unstable in oxygen-binding measurements, and the reported log p50 was calculated on the basis of fractional saturations determined under metastable conditions (data not shown). As a consequence, this data point is associated with greater uncertainty than the other data presented in this article.

Data for the kinetics of CO combination for each variant generally appear in the same article as the crystal studies except those for HbA and ({alpha}Y42A)2(ß)2 (Noble et al. 2001), for desArg Hb (Bettati et al. 1997), and for Hb Rothschild in the presence of 100 µM IHP and the absence or presence of 100 mM chloride, which are reported here for the first time.

In general these combination reactions are accelerating, the apparent rate constant increasing as the reaction proceeds. Because the appropriate comparison is between the properties of the T-state crystal and those of the deoxyHb molecule in solution in the presence of IHP, the available kinetic data were reanalyzed to estimate the rate constant for the initial reaction of CO with deoxyHb. Advantage was taken of our observation that the time course of CO combination kinetics can be well approximated by a two-step, sequential kinetic process:



Fitting the kinetic transients to the above equation yields a good estimate of the initial reaction rate constant, l`init, which is second order and equal to, or rate limited by, l`1, the rate constant for the binding of the first CO molecule to deoxyHb. It is now possible to examine how well l`init estimates l`1. Recently, the rate constants for the combination of the first CO molecule with the symmetrical FeZn hybrids of HbA and a series of variants, including ßW37E, have been reported (Noble et al. 2001). These are Hbs in which the heme groups of either both {alpha} subunits or both ß subunits have been replaced by zinc protoporphyrin IX. Because the Zn porphyrin is an excellent mimic of unliganded heme, but is unable to bind a ligand, these measurements yield the separate values of l`1 for the {alpha} and for the ß subunits. The average of the l`1 values for the two types of subunits yields an estimate of l`1 for the heme-containing tetramer. In Table 1Go, log l`1 estimated in this way is compared with log l`init for this series of Hbs in the presence of IHP. The close correspondence between log l`1 and log l`init is evident.


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  		Table 1. Comparison of log l`1, the rate contant for the binding of the first CO molecule to deoxygenated Hb, and log l`initthe rate constant for the initial binding of CO to deoxygenated Hba
 


   Acknowledgments
 
The authors acknowledge support from USPHS National Institutes of Health Program Project PO1 GM58890.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

Dedication

Max Perutz long advocated the position that the structures of proteins in crystals are relevant to the understanding of function in solution. The work reported here strongly supports this point of view, and we wish to dedicate this article to him.


   References
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 Abstract
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 Results and Discussion
 Materials and methods
 References
 
Ackers, G.K. 1998. Deciphering the molecular code of hemoglobin allostery. Adv. Protein Chem. 51: 185–253.[Medline]

Bettati, S., Kwiatkowski, L.D., Kavanaugh, J.S., Mozzarelli, A., Arnone, A., Rossi. G.L., and Noble, R.W. 1997. Structure and oxygen affinity of crystalline des-His-146ß human hemoglobin in the T state. J. Biol. Chem. 272: 33077–33084.[Abstract/Free Full Text]

Bonaventura, J., Bonaventura, C., Brunori, M., Giardina, B., Antonini, E., Bossa, F., and Wyman, J. 1974. Functional properties of carboxypeptidase-digested hemoglobins. J. Mol. Biol. 82: 499–511.[CrossRef][Medline]

Bruno, S., Bonaccio, M., Bettati, S., Rivetti, C., Viappiani, C., Abbruzzetti, S., and Mozzarelli, A. 2001. High and low oxygen affinity conformations of T state hemoglobin. Protein Sci. 10: 2401–2407.[Abstract/Free Full Text]

Doyle, M.L., Lew, G., DeYoung, A., Kwiatkowski, L., Wierzba, A., Noble, R.W., and Ackers, G.K. 1992. Functional properties of human hemoglobins synthesized from recombinant mutant ß-globins. Biochemistry 31: 8629–8639.[CrossRef][Medline]

Eaton, W.A., Hofrichter, J., Henry, E.R., and Mozzarelli, A. 1999. Is cooperative oxygen binding by hemoglobin really understood? Nat. Struct. Biol. 6: 351–358.[CrossRef][Medline]

Edelstein, S.J., Rehmar, M.J., Olson, J.S., and Gibson, Q.H. 1970. Functional aspects of the subunit association-dissociation equilibria of hemoglobin. J. Biol. Chem. 245: 4372–4381.[Abstract/Free Full Text]

Gibson, Q.H. 1959. The kinetics of reactions between haemoglobin and gases. Prog. Biophys. Biophys. Chem. 9: 1–52.

Hernan, R.A., Hui, H.L., Andracki, M.E., Noble, R.W., Sligar, S.G., Walder, J.A., and Walder, R.Y. 1992. Human hemoglobin expression in Escherichia coli: Importance of optimal codon usage. Biochemistry 31: 8619–8628.[CrossRef][Medline]

Hui, H.L., Kavanaugh, J.S., Doyle, M.L., Wierzba, A., Rogers, P.H., Arnone, A., Holt, J.M., Ackers, G.K., and Noble, R.W. 1999. Structural and functional properties of human hemoglobins reassembled after synthesis in Escherichia coli. Biochemistry 38: 1040–1049.[CrossRef][Medline]

Imai, K. 1982. Allosteric effects in hemoglobin. Cambridge University Press, Cambridge.

Imai, K. and Yonetani, T. 1975. pH dependence of the Adair constants of human hemoglobin. Nonuniform contribution of successive oxygen bindings to the alkaline Bohr effect. J. Biol. Chem. 250: 2227–2231.[Abstract/Free Full Text]

Kavanaugh, J.S., Rogers, P.H., Case, D.A., and Arnone, A. 1992. High resolution X-ray study of deoxyhemoglobin Rothschild 37ß Trp -> Arg: A mutation that creates an intersubunit chloride-binding site. Biochemistry 31: 4111–4121.[CrossRef][Medline]

Kavanaugh, J.S., Chafin, D.R., Arnone, A., Mozzarelli, A., Rivetti, C., Rossi, G.L., Kwiatkowski, L.D., and Noble, R.W. 1995. Structure and oxygen affinity of crystalline desArg141{alpha} human hemoglobin A in the T state. J. Mol. Biol. 248: 136–150.[CrossRef][Medline]

Kavanaugh, J.S., Weydert, J.A., Rogers, P.H., Arnone, A., Hui, H.L., Wierzba, A.M., Kwiatkowski, L.D., Paily, P., Noble, R.W., Bruno, A. et al. 2001. Site-directed mutations of human hemoglobin at residue 35ß: A residue at the intersection of the {alpha}1ß1, {alpha}1ß2, and {alpha}1{alpha}2 interfaces. Protein Sci.. 10: 1847–1855.[Abstract/Free Full Text]

Kilmartin, J.V. and Wootton, J.F. 1970. Inhibition of Bohr effect after removal of C-terminal histidines from haemoglobin ß chains. Nature 228: 766–767.[CrossRef][Medline]

Kilmartin, J.V., Hewitt, J.A., and Wootten. J.F. 1975. Alteration of functional properties associated with the change in quaternary structure in unliganded hemoglobin. J. Mol. Biol. 93: 203–218.[CrossRef][Medline]

Luisi, B., Liddington, R., Fermi, G., and Shibayama, N. 1990. Structure of deoxy-quaternary haemoglobin with liganded beta subunits. J. Mol. Biol. 214: 7–14.[CrossRef][Medline]

Matthew, J.B., Hanania, G.I.H., and Gurd, F.R.N. 1979. Electrostatic effects in hemoglobin: Bohr effect and ionic strength dependence of individual groups. Biochemistry 10: 1927–36.

Matthew, J.B., Friend, S.H., and Gurd, F.R.N. 1982. Electrostatic interactions associated with effector binding to hemoglobin. In Hemoglobin and oxygen binding (eds. C. Ho, W.A. Eaton, J.P. Collman, Q.H. Gibson, J.S. Leigh Jr., E. Margoliash, K. Moffat, and W.R. Scheidt), pp. 231–241. Elsevier North-Holland, New York.

McDonald, M.J., Bleichman, M., Bunn, H.F., and Noble, R.W. 1979. Functional properties of the glycosylated minor components of human adult hemoglobin. J. Biol. Chem. 254: 702–707.[Free Full Text]

Mozzarelli, A., Rivetti, C., Rossi, G.L., Henry, E.R., and Eaton, W.A. 1991. Crystals of haemoglobin with the T quaternary structure bind oxygen noncooperatively with no Bohr effect. Nature 35: 416–419.

Mozzarelli, A., Bettati, S., Rivetti, C., Rossi, G.L., Colotti, G., and Chiancone, E. 1996. Cooperative oxygen binding to Scapharca inaequivalvis hemoglobin in the crystal. J. Biol. Chem. 271: 3627–3732.[Abstract/Free Full Text]

Mozzarelli, A. and Rossi, G.L. 1996. Protein function in the crystal. Annu. Rev. Biophys. Biomol. Struct. 25: 343–365.[CrossRef][Medline]

Mozzarelli, A., Rivetti, C., Rossi, G.L., Eaton, W.A., and Henry, E.R. 1997. Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals. Protein Sci. 6: 484–489.[Abstract]

Mozzarelli, A. and Bettati, S. 2001. Functional properties of immobilized proteins. In Advanced functional molecules and polymers (ed. H. S. Nalwa), Vol. 4, pp.55–97. Gordon and Breach Science Publishers, Singapore.

Noble, R.W., Hui, H.L., Kwiatkowski, L.D., Paily, P., DeYoung, A., Wierzba, A., Colby, J.E., Bruno, S., and Mozzarelli, A. 2001. Mutational effects at the subunit interfaces of human hemoglobin: Evidence for a unique sensitivity of the T quaternary state to changes in the hinge region of the {alpha}1ß2 interface. Biochemistry 40: 12357–12368.[CrossRef][Medline]

Paoli, M., Liddington, R., Tame, J., Wilkinson, A., and Dodson, G. 1996. Crystal structure of T state haemoglobin with oxygen bound at all four haems. J. Mol. Biol. 256: 775–792.[CrossRef][Medline]

Parkhurst, L.J. and Gibson, Q.H. 1967. The reaction of carbon monoxide with horse hemoglobin in solution, in erythrocytes, and in crystals. J. Biol. Chem. 242: 5762–5770.

Pennelly, R.R. and Noble, R.W. 1978. Functional identity of hemoglobins S and A in the absence of polymerization. In Biochemical and clinical aspects of hemoglobin abnormalities (ed. W.S. Caughey), pp. 401–411. Academic Press, New York.

Poyart, C.F., Bursaux, E., and Bohn, B. 1978. An estimation of the first binding constant of O2 to human hemoglobin A. Eur. J. Biochem. 87: 75–83.[Medline]

Rivetti, C., Mozzarelli, A., Rossi, G.L., Henry, E.R., and Eaton, W.A. 1993a. Oxygen binding by single crystals of hemoglobin. Biochemistry 32: 2888–2906.[CrossRef][Medline]

Rivetti, C., Mozzarelli, A., Rossi, G. L., Kwiatkowski, L., Wierzba, A.M., and Noble, R.W. 1993b. Effect of chloride on oxygen binding to crystals of hemoglobin Rothschild (ß37 Trp -> Arg) in the T quaternary state. Biochemistry 32: 6411–6418.[CrossRef][Medline]

Russo, R., Benazzi, L., and Perrella, M. 2001. The Bohr effect of hemoglobin intermediates and the role of salt bridges in the tertiary/quaternary transitions. J. Biol. Chem. 276: 13628–13634.[Abstract/Free Full Text]

Schreiber, J.K. and Parkhurst, L.J. 1984. Ligand binding equilibrium and kinetic measurements on the dimeric myoglobin of Busycon canaliculatum and the comparative ligand binding of diverse non-cooperative heme proteins. Comp. Biochem. Physiol. 78A: 129–135.[CrossRef]

Sun, D.P., Zou, M., Ho, N.T., and Ho. C. 1997. Contribution of surface histidyl residues in the {alpha}-chain to the Bohr effect of human normal adult hemoglobin: Roles of global electrostatic effects. Biochemistry 36: 6663–6673.[CrossRef][Medline]

Tan A.L., Noble, R.W., and Gibson, Q.H. 1973. Conditions restricting allosteric transitions in carp hemoglobin. J. Biol. Chem. 248: 2880–2888.[Abstract/Free Full Text]


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