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[11] Study of the Bohr effect in hemoglobin intermediates
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[ 1 1] S t u d y o f t h e B o h r E f f e c t i n Hemoglobin Intermediates
By
LOUISE BENAZZI, R O S A R I A RUSSO, M A R I L E N A RIPAMONTI, and MICHELE PERRELLA
Introduction Allosteric proteins are devices that transduce signals across a molecule by means of conformational changes. Because allosteric proteins carry out key cellular functions, the mechanisms by which such intramolecular signaling occurs are one of the most intriguing and challenging areas of biophysical research. The tertiary structural changes that underlie the allosteric phenomenon trigger quaternary structural changes in multimeric cooperative proteins. In these complex systems, one type of ligand, the homotropic ligand, binds the subunits cooperatively while the assembly of subunits switches from a low-affinity T to a high-affinity R quaternary conformation. Another type of ligand, the heterotropic ligand, by allosteric binding the subunits modulates the affinity for the homotropic ligand. The thermodynamics of homo- and heterotropic interactions can be a powerful tool for the study of intramolecular signaling provided that the intermediate states of ligation of the protein are accessible to investigation. Hemoglobin is ideally suited for such studies. The intermediate states of ligation with oxygen are not accessible to investigation for technical reasons, but a variety of alternative ligands are available. In landmark studies carried out using the cyanide complex of the ferric subunit, 1-4 extended later to the metal substituted hemoglobins as models of ligation, 5-7 Ackers and collaborators have discovered that the intermediate states of ligation are grouped into three distinct energetic levels. The lowest level corresponds to deoxy hemoglobin, species [01] in Fig. 1. The two monoliganded and one diliganded intermediate, species [11], [12], and [21], form the median level. The remaining species, [22], [23], [24], [31], and [32], belong to the highest level 1 F. R. Smith and G. K. Ackers, Proc. NatL Acad. Sci. U.S.A. 82, 5347 (1985). 2 M. Perrella, L. Benazzi, M. A. Shea, and G. K. Ackers, Biophys. Chem. 35, 97 (1990). 3 M. A. Daugherty, M. A, Shea, J. A. Johnson, V. J. LiCata, G. L. Turner, and G. K. Ackers, Proc. Natl. Acad. Sci. U.S.A. 88, 1110 (1991). 4 M. L. Doyle and G. K. Ackers, Biochemistry 31, 11182 (1992). 5 G. K. Ackers and F. R. Smith, Annu. Rev. Biophys. Chem. 16, 583 (1987). 6 F. R. Smith, D. Gingrich, B. M. Hoffrnan, and G. K. Ackers, Proc. Natl. Acad. Sci. U.S.A. 84, 7089 (1987). 7 p. C. Speros, V. J. LiCata, T. Yonetani, and G. K. Ackers, Biochemistry 30, 7254 (1991).
METHODS IN ENZYMOLOGY, VOL. 295
Copyright © 1998by AcademicPress All rights of reproduction in any form reserved. 0076-6879/98 $25.00
[11]
BOHR EFFECT IN HEMOGLOBIN INTERMEDIATES
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Ol
(~) (~[~)
11
12
(~'[~)(a[~)
21
(~') (~[3)
22
23
31
24
32
41
(a~.[~.) (a~.[~L) FIG. 1. The 10 ligation states of hemoglobin. The ligand, L, can be 02, CO, NO, or the complex of CN with the heine in the ferric state.
of fully liganded hemoglobin, species [41]. Thus the energies are not distributed into discrete sets corresponding to each ligation state and the two species in which one o~ and one/3 subunit are liganded, i.e., species [21] and [22], are energetically nonequivalent. The inconsistency of this finding with a concerted model of cooperativity s has been the main argument in favor of an alternative mechanism proposed by Ackers, known as the "symmetry rule. ''9 Information on the properties of the intermediates has also been gained by the study of the interaction of hemoglobin with carbon monoxide, both at equilibrium and under dynamic conditions. 1°-12 The large difference in the energetic properties of intermediates [21] and [22] observed in the 8 j. Monod, J. Wyman, and J. P. Changeaux, J. Mol. Biol. 12, 88 (1965). 9 G. K. Ackers, M. L. Doyle, D. M. Myers, and M. A. Daugherty, Science 255, 54 (1992). ,0 M. Perrella, L. Sabbioneda, M. Samaja, and L. Rossi-Bernardi, J. Biol. Chem. 261, 8391 (1986). ~1 M. Perrella, A. Colosimo, L. Benazzi, M. Ripamonti, and L. Rossi-Bernardi, Biophys. Chem. 37, 211 (1990). 12 M. Perrella, N. Davids, and L. Rossi-Bernardi, J. Biol. Chem. 267, 8744 (1992).
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cyanomet model was not confirmed by the CO equilibrium studies. H,~3This apparent discrepancy has been used to refute the general validity of the symmetry rule. 14 This criticism neglects the fact that the mechanism of hemoglobin function, with respect to both ligand affinity and cooperativity, is quantitatively modulated by environmental conditions. The CO model simulates the highly cooperative behavior of oxygen. It apparently conforms to the predictions of the concerted model of cooperativity, as under physiological conditions most of the energy changes in ligand binding that determine cooperativity arise from quaternary state transitions. In contrast, the energetics of the deoxy/cyanomethemoglobin model of ligation indicates that a significant fraction of the energy change in ligand binding arises from tertiary state transitions within the T quaternary state. 9 The study of the association reaction between hemoglobin and CO under nonphysiological conditions, such as high pH, also suggests that the interactions of CO can generate significant cooperativity by means of tertiary structural changes within the T state, is Thus what appears to be a contradiction between ligation models turns out to be a powerful means of investigation into the mechanism, as a wide spectrum of functional properties becomes accessible to measurement as a result of changes in type of ligand or environmental conditions. Bohr Effect of Hemoglobin The binding of oxygen to hemoglobin releases protons. This phenomenon, known as the Bohr effect, occurs with any type of ligand and also on oxidation of the ferrous hemes of the protein. It is due to changes in the pK value of charged groups in response to the conformational changes undergone by the protein in the course of ligand binding. Although the nature of the charged groups is partly a matter of controversy, it is accepted that these groups are located on the a and/3 subunits and that the changes in pK result both from salt bridge and hydrogen bond interactions and from the interactions of certain ions, such as chloride, with charged groups of the protein. 16 Ackers and collaborators have studied the effects on the energetics of the deoxy/cyanomet model of ligation of several heterotropic ligands, including proton, and have shown that the heterotropic ligand does not modify the quaternary state of the protein.t7 Consistent with the energetics 13 M. Perrella and I. Denisov, Methods Enzymol. 259, 468 (1995). 14 S. J. Edelstein, J. Mol. Biol. 257, 737 (1996). 15 C. Ho, Biochemistry 26, 6299 (1987). 16 A. Arnone, R. E. Benesch, and R, Benesch, J. Mol. Biol. 115, 627 (1977). 17 A. Daugherty, M. A. Shea, and G. K. Ackers, Biochemistry 33, 10345 (1994).
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is the observation reported by Perrella et al.18that the Bohr protons released by the intermediates in the deoxy/cyanomet system are due to perturbations of the tertiary structures of the subunits within each quaternary structure. Thus the energetics of the intermediate states of ligation in the deoxy/ cyanomet model provides a thermodynamic probe of the tertiary and quaternary structures of the protein, and the Bohr protons released at each ligation state probe the tertiary structures of the liganded subunits in each quaternary state. The information from the two types of thermodynamic studies can provide a means of tracking the pathways of intersubunit communication. Before we describe the methodology for the measurement of the Bohr effect of the intermediates and how this strategy can be potentiated further, the following briefly describes the results of our previous study of the Bohr effects of the deoxy/cyanomethemoglobin intermediates.
Bohr Effects of Hemoglobin Intermediates
Definition of Bohr Effect of Cyanomet Intermediates The protons released on changing a ferrous subunit into a cyanomet subunit cannot be measured directly. Bohr effect of a cyanomet intermediate is described as the difference between the total Bohr effect of hemoglobin at a certain pH value and the protons released by exposing to oxygen the unliganded subunits of the intermediate at the same pH.
Using Deoxy/Cyanomet Model of Ligation The cyanomet model of ligation solves the problem of the mobility of the homotropic ligand that prevents the study of the oxygen intermediates. The high affinity for cyanide of the ferric subunits, as compared with the ferrous subunits, immobilizes cyanide onto these subunits. The rationale for assuming such a complex as a model of ligation is that the crystal structure of cyanomethemoglobin is similar to that of aquomethemoglobin, which is similar to the structure of oxyhemoglobin. However, contrary to aquomethemoglobin, the spin state of the ferric iron of cyanomethemoglobin is low as that of the ferrous ion in oxyhemoglobin.19-22 ~s M. Perrella, L. Benazzi, M. Ripamonti, and L. Rossi-Bernardi, Biochemistry 33, 10358 (1994). 19 M. F. Perutz and F. S. Mathews, J. Mol. Biol. 21, 199 (1966). 20 M. F. Perutz, H. Muirhead, J. M. Cox, L. C. G. Goaman, F. S. Mathews, E. L. McGandy, and L. E. Webb, Nature 219, 29 (1968). 21 M. F. Perutz, H. Muirhead, J. M. Cox, and L. C. G. Goaman, Nature 219, 131 (1968). 22 E. J. Heidner, R. C. Ladner, and M. F. Perutz, J. Mol. Biol. 104, 707 (1976).
zNrSaGZVICSOF BIOLOGICALMACROMOLECULES
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[1 1]
Only those intermediates that dissociate into identical dimers, such as species [23] and [24], can be studied in a pure form. Intermediates that dissociate into different dimers, indicated in the following as hybrids, such as species [21], [22], [31], and [32], disproportionate rapidly into their parental species, as depicted in Fig. 2. Thus hybrids can be studied under physiological conditions only in the presence of their parental species.
Determination of Bohr Effect of Cyanomet Intermediates The Bohr effects of hemoglobin and of intermediates [23] and [24] are measured using the pure species prepared as in the Experimental Procedures. To determine the Bohr effect of a hybrid species, the protons released by exposing the anaerobic equilibrium mixture of the hybrid and parental species to oxygen are measured. The amount of protons released by the vacant sites of the hybrid species can be calculated if the Bohr effect of the pure parental species and the fraction of each parental species in the mixture are known. The Bohr protons released by the pure hybrid, H+Hyb, are then calculated as follows: H+Hyb = (H+Mix -- H+ParlfParl
-
H+Par2fPar2)(1/fHyb)
(1)
where H+Mix, H+parl, and H+r,ar2 are the protons/tetramer released by the mixture containing parental species and hybrid, and by each of the parental species. The molar fractions of the hybrid and parental species at equilibrium a r e fHyb, fParl, and fPar2" To measure the protons/tetramer, H+Mix, H+Parl, and H+ear2, a known amount of the pure parental species or mixture is deoxygenated, the pH at equilibrium is measured, and the protons released on oxygenation are .... ~
~ . . . . ~'
~ . . . . . ~'
II
[I
If
II
[I
I~. . . .
~
~ ....
~L
6" . . . .
Parent A A
Hybrid AB
II Dimer A
Parent
II ~'
BB
II
aL Dimer B
FIG. 2. Scheme of the dimer exchange reactions between parent species AA (e.g., deoxyhemoglobin) and parent species BB (e.g., cyanomethemoglobin) yielding hybrid species AB and, conversely, of the disproportionation reaction of hybrid AB to yield parents AA and BB. Dashed lines indicate the main intersubunit contacts that break on dimerization.
[1 l]
213
BOHR EFFECT IN HEMOGLOBIN INTERMEDIATES
titrated with NaOH. The molar fractions of the species in equilibrium are determined by cryogenic techniques as described in the Experimental Procedures. The Bohr effect of a parental or hybrid species, B.e.int, is (2)
B.e.int = B.e.Hb -- H+int
where B.e.Hb is the total Bohr effect of hemoglobin and H+int is the amount of protons released on oxygenation of the vacant sites of the intermediates.
Bohr Effects versus pH of Cyanomet Intermediates Three typical curves for the cyanomet intermediates are obtained by measuring the Bohr protons released at different pH values. These pH profiles are shown in Fig. 3 together with the bell-shaped curve of the total Bohr effect of hemoglobin in the same pH range, pH 6.2-9. The difference between the total Bohr effect of hemoglobin and the Bohr effect of a liganded species at the same pH value measures the Bohr protons that are released by the unliganded subunits of that species on oxygenation. Monoliganded Intermediates [11] and [12]. A bell-shaped curve characterizes the pH profiles of the Bohr effect of the monoliganded species [11] and [12] (Fig. 3A), which maintain the T quaternary state at all pH values according to the energetics. 17 Ligation of the c~ or/3 subunit modifies the pK of one or more charged groups, resulting in the release of protons. The pK modification is such that the interactions involved are disrupted at a pH value, in the range of pH 8-8.5, significantly lower than that at which 2.5
B
01
2.0
1,5 ",...
1,0 0.5 0,0 6.5 7 . 0 7 . 5 8.0 8.5
6.5 7.0 7 . 5 8.0 8.5
6.5 7=0 7.5 8.0 8.5
pH
pH
pH
FIG. 3. Bohr effect (B.c.) of intermediates versus pH. (A) Total Bohr effect of Hb, species [01] [also shown in (B) and (C)], and B.e. of species [11] and [12]; (B) B.e. of species [23], [24], [31], and [32]; (C) B.e. of species [21], [22], and calculated sum of species [11] and [12] and B.e. of [21] multiplied by 2. Lines were obtained by fitting experimental data TM using polynomials.
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the same groups cease to release protons in hemoglobin (about pH 9.5). The Bohr effect of the unliganded subunits is maintained at pH 8-8.5, in full or in part, despite the possible structural perturbations induced in the protein by ligation of one subunit. Diliganded Intermediates [22], [23], and [24]. These species, which are in the R quaternary state according to the energetics, yield pH profiles of the Bohr effect almost sigmoidal in shape, as in Fig. 3B. At pH 8-8.5, the amount of Bohr protons released by these species equals the total Bohr effect of hemoglobin. This indicates that the liganded and unliganded subunits have the same quaternary and tertiary structures of the subunits of oxyhemoglobin. At neutral and acidic pH, these species release an amount of Bohr protons comparable to that released by monoligation. Under these conditions the amount of protons released by oxygenation of the two unliganded subunits of species [22], [23], and [24] is almost the same as that released by oxygenation of three unliganded subunits in species [11] and [12]. Because species [22], [23], and [24] are in the R quaternary state, 17 pH must induce modifications of the tertiary structures of the unliganded subunits in the R quaternary state, which in releasing Bohr protons mimic the tertiary structures of the subunits in the T quaternary state. Diliganded Intermediate [21]. Ackers and collaborators have found that the change in energy due to cooperativity is the same for the binding of one ligand as for the binding of two ligands yielding the configuration of intermediate [21]. 9 This indicates that, along the pathway in ligand binding centered on intermediate [21], which maintains the T quaternary state, cooperativity arises from changes in tertiary structure of the dimeric halfmolecule. In contrast, according to the concerted model, cooperativity should arise from the increase in the population of molecules in the R quaternary state brought about by the increase in the number of bound ligands. 8 The interpretation of the energetics is entirely consistent with the pH profile of the Bohr effect of species [21] (Fig. 3C). It is not sigmoidal in shape as the profile of the diliganded species [22], which is in the R quaternary state and also liganded at one o~ and one/3 subunit. It is bell shaped as the profile of the monoliganded species, but it is equal neither to the sum of the pH profiles of the two monoliganded species nor to onehalf the total Bohr effect of hemoglobin. This finding is consistent with the energetics, as it indicates that interactions occur in the T quaternary state between the tertiary structures of the subunits of the dimer. Triliganded Species [31] and [32]. The pH profiles of the Bohr effects of these species, which are in the R quaternary state according to the energetics, are also almost sigmoidal in shape as those of the diliganded species [22], [23], and [24] (Fig. 3B) and are interpreted similarly.
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215
Comparison with Oxygen Ligation The Bohr protons released on changing a ferrous into a cyanomet subunit are calculated using the oxygen Bohr effect of the vacant sites. The validity of this procedure is supported by the observation that at pH 7.4 the calculated increments in Bohr effect at each step of the cyanomet ligation along the various pathways in Fig. 1 add up to the total value of the oxygenation Bohr effect of hemoglobin. 18 Furthermore, at pH 7.4 the Bohr effect of the monoliganded cyanomet species, 0.62/0.59 mol H+/ tetramer, is the same, within error, as that (0.64 H+/tetramer) calculated for oxygen under similar conditions.23The overall Bohr effect for the transition from monoligation to the triliganded cyanomet state (0.95-1.4 mol H+/tetramer, depending on the pathway) 18 is not far from the value calculated for oxygen (1.62 _+ 0.27 mol H+/tetramer). 23
Bohr Effect of Mutant Hemoglobins The study of the Bohr effect of the hemoglobin intermediates indicates that a subunit uses the structural changes brought about by ligation to send across the molecules two types of signals. One signal, transmitted to the unliganded subunit across the monomer interface of the dimer, regulates the amount of energy change that occurs within the T quaternary state on binding the second subunit of the dimer. Such a change is slight or negligible in the deoxy/cyanomet model, but is more significant in the CO ligation system. 13The tertiary structural change brought about by diligation in species [21] is not the same as that brought about by monoligation as, as noted earlier, the Bohr effect of species [21] is not equivalent to the sum of the Bohr effect of the two monoliganded species. The other type of signal is sent by the ligated subunit across the dimer interface. Ligation of any one of the subunits of the adjacent dimer promotes the transition from the T to the R quaternary state, as stated by the "symmetry rule." A similar signal is sent across the dimer interface by the diliganded species [21]. Ackers and collaborators have studied the energetics of mutant hemoglobins under oxygenated and deoxygenated conditions to map the intramolecular lines of communication of the subunits interacting with the heme ligand.24,25The combination of the energetics of the intermediates of mutant hemoglobins as a function of pH and of the protons released by the Bohr 23 A. H. Chu, B. W. Turner, and G. K. Ackers, Biochemistry 23, 604 (1984). 24 G. K. Ackers and F. R. Smith, Annu. Rev. Biochem. 54, 597 (1985). 25 V. J. Licata, P. M. Dalessio, and G. K. Ackers, Prot. Struct. Funct. Genet. 17, 279 (1993).
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effect of these hemoglobins can be a powerful tool for tracking the pathways of communication across the monomer and dimer interfaces and to clarify the role of the intra- and interchain salt bridges in the quaternary state transition. A serious limitation to such studies is posed by the large amount of protein required by the measurement of the Bohr effect. The following section describes the basic technique used to obtain data summarized earlier and the modifications that could make the study of mutants feasible.
Experimental Procedures
Preparation of Hemoglobin As described later, pH measurements can be carried out on small samples of protein (60-150/~1). However, the precision of the measurements depends on the amount of protons to be determined, which in turn depends on the concentration of the protein sample. Thus methods for the rapid preparation of large quantities of purified hemoglobin and hemoglobin derivatives are a crucial step of the experimental procedure. Hemoglobin A0 is obtained by chromatography on CM-cellulose equilibrated at 4° with 5 mM potassium phosphate, 0.5 mM EDTA, pH 6.8, at 20°. Elution is carried out with 7.5 mM potassium phosphate, 0.5 mM EDTA, pH 7.33, at 20°. Using an 8 × 30-cm bed column, 4 g of hemoglobin from lysed cells yields about 2.8 g of 8 mM (heine concentration) hemoglobin A0 free from minor components as tested by isoelectric focusing (IEF) on a gel plate. The methemoglobin content is about 1.7% according to the cyanomethemoglobin method. 26 The two parental species [23] and [24] in oxyform, i.e., a2+CN-/32 and o~zfl2+cN-, are prepared as follows. Oxyhemoglobin from a blood lysate is oxidized with 1/2 equivalent of ferricyanide for 5 min at 4 ° to yield a mixture of approximately 25% each of HbO2, Hb +, ot2°2f12+, and a2÷/~2%. The mixture is equilibrated by gel filtration with 5 mM potassium phosphate, 0.5 mM EDTA, pH 6.8, at 20° and loaded on a CM-cellulose column equilibrated with the same solvent. Elution is carried out at 4° with 7.5 mM potassium phosphate, 0.5 mM EDTA, pH 7.5, at 20°. Using an 8 × 40-cm bed column loaded with 4 g of hemoglobin and eluted at 300 ml/hr, a complete resolution of species a2°2/~2+ and o~z+/32°2 is achieved in 16-20 hr. The resin portions containing these derivatives are removed by extrusion of the resin bed from the column and suspended in 20 mM Tris-HC1, 50 mM KC1, 1 mM KCN buffer, pH 7.5, at 20° to release the protein. After equilibration in 0.2 M z6K. A. Evelynand H. T. MaUoy,J. Biot Chem. 126, 655 (1938).
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KC1, 1 mM KCN, and concentration, about 0.75 g of 8 mM a2+CN-/~202 and 0.50 g of 8 mM OL202/~2+CN- are obtained. The spectrophotometric analysis of the derivatives yields the following proportions of cyanomethemoglobin: species o/2+CN-~3202, 58.0 q- 1.5 (%); species a2°2/32+cN-, 47.5 +_ 1.0 (%). The use of a 50% oxidized sample of purified hemoglobin A0 does not improve these results, which are similar to those obtained by the alternative, but more laborious, procedure of chain separation and recombination. Because intermediates [23] and [24] are key species for the preparation of all the other intermediates, the procedure for their preparation is crucial for the study of the Bohr effect of the intermediates of mutant hemoglobins. Although our procedure is simple and rapid, it may appear to be wasteful of precious material. However, if the oxy- and methemoglobin fractions, recovered from the column after chromatography, are incubated with trace amounts of ferrocyanide, they reequilibrate with the two 50% oxidized species in a few minutes. In principle, by multiple recycling, all the hemoglobin sample can be used to prepare intermediates [23] and [24].
Preparation of Samples under Anaerobic Conditions The anaerobic equilibration of the intermediates, either pure or in equilibrium with the hybrid species, can be carried out by removing oxygen enzymatically.27 Alternatively, the hemoglobin samples, titrated to a certain pH value, equilibrated, if required, with a concentration of chloride enough to saturate the chloride binding sites and containing an excess of cyanide, are deoxygenated by nitrogen tonometry and placed in glass vials sealed with a silicon cap under a positive nitrogen pressure. The vials are placed in a stoppered cylinder filled with water made oxygen free by the addition of some dithionite. The cylinder is then thermostatted in a water bath. 2 Another procedure, more convenient for the management of a large number of samples, is to connect several capped vials with each other and with a source of humidified nitrogen. The vials are thermostatted in a water bath and a slow stream of nitrogen is maintained during the time of equilibration. 18 The time required by the mixture of parental species to attain equilibrium with the hybrid under anaerobic conditions varies from 24 to 70 hr, depending on the rate of dissociation into dimers of the various tetrameric molecules, which is a function of the state of ligation of the tetramer and of pH. Excess cyanide is needed to make sure that the ferric subunits are saturated by the ligand under all conditions and throughout the equilibra-
27 V. J. LiCata, P. Speros, E. Rovida, and G. K. Ackers,
Biochemistry 29,
9771 (1990).
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ENERGETICS OF BIOLOGICAL MACROMOLECULES
I111
tion time. A significant loss of cyanide by evaporation in the experiments in which the anaerobicity is maintained by a nitrogen stream could expose the solvent to the ferric subunits facilitating electron transfer reactionsY These reactions are rapid under aerobic conditions and in the presence of ox/redox substances such as ferrocyanide, slow under anaerobic conditions, and negligible, within the time limits of the equilibration experiments, if the met subunits are kept saturated with the ligand. Thus the excess cyanide must be calibrated to the conditions of the experiments. A simple check of the stability of the cyanomet derivatives is to determine the visible spectrum of a sample of cyanomet hemoglobin before and after periods of equilibration under the same conditions of the experiments. As described later, the cryogenic method of analysis of the composition of the mixture of parental and hybrid species provides a "built-in" control of the stability of the cyanomet derivatives at the end of the equilibration. 29
Analysis of Mixtures of Parental Species and Hybrid The composition at equilibrium of the mixture can be calculated if the values of the free energies of dimer assembly into tetramers of the three species are determined using the rate constants of the association and dissociation reactions. The composition of the mixture can be determined directly under the experimental conditions of pH and ionic strength using cryogenic electrophoretic techniques. 3° The aqueous anaerobic mixture of the parent species and their hybrid is injected into a stirred aerobic cryosolvent to attain rapidly a temperature at which the tetramer dissociation reactions are so slow to render the tetramers stable for the time required by the cryogenic separation method. At - 2 5 ° the hybrids of hemoglobins A0 and S are stable for the time, about 20 hr, required to attain the equilibrium in IEF. Hybrids that are unstable under these conditions may be stabilized at lower temperatures. Separation of hemoglobins A0, S, and their hybrid can be carried out at - 4 0 ° by electrophoresis, obtaining resolution of the mixture within 3 hr. 31 Cryogenic IEF and electrophoresis are carried out on tubes of gels made of copolymers of acrylamide and methyl or ethyl acrylate using methylenebisacrylamide as a crosslinker. The higher the proportion of acrylate, the lower the temperature at which the separation can be carried out. Methyl acrylate is the reagent of choice for the lowest temperatures because 28 N. Shibayama, H. Morimoto, and S. Saigo, Biochemistry 36, 4375 (1997). ~9 G. K. Ackers, M. Perrella, J. M. Holt, I. Denisov, and Y. Huang, Biochemistry 36, 10822 (1997). 30 M. Perrella and L. Rossi-Bernardi, Methods Enzymol. 76, 133 (1981). 31 M. Perrella, M. Samaja, and L. Rossi-Bernardi, J. Biol. Chem. 254, 8748 (1979).
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it is more soluble than ethyl acrylate in the methanol-ethylene glycol-water mixtures used as the cryosolvent. Details of the preparation of the gel tubes, procedures for IEF and electrophoresis, and experiments designed to test the equilibration time of the mixtures of parent species and hybrid and to measure the equilibrium composition of the mixture have been published.13.30,32 Cryogenic separations by IEF have the additional advantage that a significant loss of cyanide from the cyanomet subunits can be detected easily, as the fractional charge due to the exposed ferric subunit of a species that has lost its bound cyanide changes its isoelectric point. Partially oxidized carboxyhemoglobin A0 yields nine resolved species by cryofocusing, whereas upon the addition of cyanide to the sample a single component is observed. 32 Thus, cryofocusing provides at the same time a means of resolving the parental and hybrid species and of checking the stability of the cyanomet derivatives during the long incubation time required for the equilibration of the mixture under anaerobic conditions. The quantitative analysis of the protein zones separated by cryogenic IEF or electrophoresis can be carried out by slicing the gels in correspondence to the isolated components, eluting the protein from the gel, and assaying the heme by the pyridine hemochromogen methodfl The total amount of protein sample required by this procedure is about 0.5 mg. Another procedure set up by Ackers and collaborators, requiring even less protein, scans the isolated colored zones of the gel tube using spectrophotometry in the visible range. 27 The cryogenic separation methods can only be used in cases where the parental and hybrid species differ in charge or isoelectric point and the hemoglobins are stable under the conditions of the cryosolvent required by the technique. If the species under study is the hybrid of hemoglobin A0 and a mutant or chemically modified hemoglobin A0 not significantly different in charge from hemoglobin A0, the cryogenic separation of the species can be carried out by replacing hemoglobin A0 with, e.g., hemoglobin S (Glu6/3 ~ Val) or C(Glu6/3 --~ Lys), which differ in charge but are identical to hemoglobin A0 with respect to all other functional properties.
Measurements of pH The measurement of the pH of the samples under anaerobic conditions and the titration with NaOH of the protons released by oxygenation can be carried out using the equipment shown in Fig. 4. Because the pH value of the oxygenated hemoglobin sample is backtitrated to the value of the 32 M. Perrel|a and L. Rossi-Bernardi, Methods Enzymol. 232, 445 (1994).
220
ENERGETICS
OF BIOLOGICAL
MACROMOLECULES
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~ - 6mm ~ [
¢*m~--[
h-
--fl t ~ Carom
.*
d"
["
.t,
1Sin
6
5
3
~4 2 7 F]G. 4. Titration vessel: 1, water-jacketed combined calomel-glass electrode; 2, waterjacketed vessel; 3, vessel cover; 4, nitrogen inlet; 5, oxygen inlet; 6, gas outlet; 7, magnetic bar (adapted from Perrella et al.tS). (Inset) Vessel for pH determination on 60-/.1 samples.
initial a n a e r o b i c c o n d i t i o n s , t i t r a t i o n can b e a v o i d e d if t h e b u f f e r c a p a c i t y o f o x y h e m o g l o b i n as a f u n c t i o n o f p H is k n o w n . T h e b u f f e r c a p a c i t y o f o x y h e m o g l o b i n , B, is d e f i n e d as B = aOH-/ApH[HbO2]
(3)
[ 1 1]
BOHREFFECTIN HEMOGLOBININTERMEDIATES
221
where AOH- are the equivalents of NaOH used for the titration, ApH is the change in pH from the anaerobic to the aerobic conditions, and [HbO2] is the oxyhemoglobin heme concentration. The Bohr protons released by oxygenation of the vacant sites of the intermediates can be calculated as follows: H + (per tetrameric hemoglobin) = 4BApH (4) where 4 is a factor required if the oxyhemoglobin concentration is measured on a heme basis. The apparatus in Fig. 4 was used to titrate with 20 mM NaOH after exposure to oxygen samples of deoxyhemoglobin (1 ml) in the pH range 6.2-9.18 Data on the Bohr effect obtained by this procedure are shown in Fig. 5A. The buffer capacity of oxyhemoglobin calculated from the hpH and zXOH- values recorded in such experiments is shown in Fig. 5B. The Bohr effect of species [11], [23], and [31] calculated from the buffer capacity of oxyhemoglobin and the ApH values measured by exposing to oxygen the pure parental species or their equilibrium mixtures are checked in Fig. 6 against experimental data obtained by the titration method. 18 Clearly, the pH profiles of the Bohr effect of the intermediates can be obtained with the same precision either by titration or by the buffer capacity approach. Such an approach is based on just two pH determinations, under anaerobic and aerobic conditions, and the volume of sample required depends on the technique used for the measurement of pH.
Measurements of p H on Small Samples Combined Electrode Method. The equipment shown in Fig. 4 can be adapted to measure the pH in 60-/xl samples using the device shown in the insert. A glass tube, 4 mm i.d. and 1.5 cm long, is mounted vertically on a Teflon stand, which is steadily rotated inside the thermostatted vessel of Fig. 4 by means of a magnet inserted in the base. The pH of the hemoglobin sample is measured using a combined microelectrode (Ingold INLAG 423, Ag[AgC1, Mettler-Toledo AG, Switzerland). Figure 7 shows the time required to reach a stable pH reading in a solution of deoxyhemoglobin and the pH change observed on oxygenation of this solution using such a device. The values of the Bohr effect of hemoglobin measured by the buffer capacity approach using this method are comparable to those obtained by the titration method (Fig. 5). DifferentialpH Method. Figure 8 shows the scheme of a differential pH m e t e r y which is a development of an earlier apparatus, 34 adapted for 33 Patent applied to U.K. Patent Office by University of Milan on May 2, 1996 (ref. 9609249-9). 34 M. Luzzana, G. Dossi, A. Mosca, A. Granelli, D. Berger, E. Rovida, M. Ripamonti, A. Musetti, and L. Rossi-Bernardi, Clin. Chem. 29, 80 (1983).
222
[11]
ENERGETICS OF BIOLOGICAL MACROMOLECULES
A 2.5
2.0 o E
1.5
1.0
% 0.5 0 0
B
I
0.0
~
I
3.5
~o
3.0
•
0
0
o
2.5 0
~E ::t. 2 . 0
v
-r" v
1.5
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1.0 0.5 0.0 6.0
6.5
I
i
7.0
7.5
1
8.0
I
8.5
9.0
pH
FIG. 5. (A) Protons/tetramer released on oxygen exposure of unliganded subunits of species [01] (O) (adapted from Perella eta/. TM)and calculated Bohr effect of species [01] ([~) using the buffer capacity approach and the vessel shown in the inset of Fig. 4 to measure ~pH values. (B) Buffer capacity of oxyHb (O) calculated from ApH and AOH- values recorded in experiments for the determination of Bohr effect of species [01] shown in A, and buffer capacity of oxyHb S (V).
[11]
BOHR EFFECT IN HEMOGLOBIN INTERMEDIATES
223
2.5
A 2.0 1.5 1.0 0.5
B 2.0
;:1=
1.5 1.0
0 0.5
C 2.0 1.5 1.0 0.5 0.0 5.0
i
I
i
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i
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7.0
7.5
8.0
8.5
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Fl~. 6. C o m p a r i s o n of the Bohr effect of intermediates versus p H as determined by titration experiments (adapted from Perrella e t aLaS), and Bohr effect calculated using the buffer capacity of oxyHb. [The total Bohr effect of species [01] is shown in ( A ) - ( C ) as a reference.] Experimental (O) and calculated ( - ) Bohr effects of the hybrid are indicated in each panel (the two lines indicate the error band width).
preliminary experiments using concentrated solutions of oxyhemoglobin (10 g/dl) at different pH values. Two capillary glass electrodes (6 tzl inner volume) connected by a loop (90-100/A inner volume) measure the difference in pH between the solutions filling the electrodes. Solutions can be drawn into the system, or driven out, by means of micropumps or, in the modified apparatus shown in Fig. 8, by a gas-tight syringe connected to the outlet of one electrode (electrode B). The other electrode (electrode A) is connected to a reservoir. The total inner volume of the electrodes, loop, and connections to the syringe and reservoir is about 150 ~1. The experi-
224
7.7 Q
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7.6
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0
t
,
0
0
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I
i 8
J 9
i 10
11
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ENERGETICS OF BIOLOGICAL MACROMOLECULES
0
7
©
7.5
7.4
' 0
I
2
,
I
4
,
I
6
,
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,
0
0
I
,
12
0
0
I
,
14
0 I
16
i
I
18
;
20
t(min) FI~. 7. Time required to reach the electrode equilibrium before and after oxygenation of the sample (60/xl) of concentrated hemoglobin solution (10 g/dl) contained in the vessel shown in the insert of Fig. 4. (Inset) Precision of the pH readings.
ments were aimed at determining the sensitivity, stability, and reproducibility of the differential pH measurements and the volume of protein solution required to replace completely the solution contained in one capillary electrode with a solution of different pH.
FIG. 8. Scheme of a differential pH meter. It is composed of two glass microelectrodes (A and B) connected by a loop (L). A gas-tight syringe (S) connected to the outlet of electrode B draws the solutions into and out of the electrodes; electrode A is connected to a reservoir (R).
[11]
225
BOHR EFFECT IN HEMOGLOBIN INTERMEDIATES 150
100
50 0
~<-~-
x
............... .
-50
•
--~'- ........
2
\.A
-100
-150
'
0
50
I I' 200 sarnp]e v o l u m e
' 250
300
(/~I)
FIG. 9. Cyclic measurements of the difference in pH between concentrated solutions of oxyhemoglobin (10 g/dl) using the differential pH meter of Fig. 8. Initial conditions: both capillary electrodes are in equilibrium with the same solution at pH 7.0 (point 1). A solution at pH 6.9 is drawn into the system. The initial solution is displaced completely from electrode A by 90-100 /zl of solution at pH 6.9 drawn into the system (point 2). The maximal pH difference between electrode A (pH 6.9) and electrode B (pH 7.0) is recorded at point 2. More solution at pH 6.9 is drawn in to replace the solution at pH 7.0, filling electrode B (point 3). The solution at pH 7.0 is drawn in to replace the solution at pH 6.9, filling electrode A (90-100/zl). A difference in pH of equal magnitude and inverted in sign as shown in point 2 is recorded at point 4.
In the experiment depicted in Fig. 9, the system was filled with a solution of oxyhemoglobin at pH 7.0. A pH difference between the electrodes due to the slightly different signals from the two electrodes in equilibrium with the same solution was zeroed. By acting on the syringe, a solution of oxyhemoglobin of the same concentration at pH 6.9 was then drawn into electrode A (point 1 in Fig. 9) at 10-/zl steps to replace the previous solution at pH 7.0 and at each step the pH was recorded. At the same time, identical amounts of solution at pH 7.0 contained in the loop moved into electrode B. As shown in Fig. 9, the maximal pH difference between the electrodes was measured after drawing in 80-90/~1 of solution at pH 6.9 (point 2). This amount was the minimum volume of solution of oxyhemoglobin at pH 6.9 required to replace the solution at pH 7.0 in electrode A and in
226
E N E R G E T I C S OF B I O L O G I C A L M A C R O M O L E C U L E S
[11]
the tube connecting it to the reservoir under conditions of nonturbulent flow. Because the inner volume of the loop was 90-100/zl, electrode B was still in equilibrium with the initial solution at pH 7.0, which was drawn in from the loop. More solution at pH 6.9 was drawn in to replace the solution at pH 7.0 filling electrode B. The pH difference between the electrodes, inverted in sign, decreased in magnitude until a ApH of _+0.001 was reached again when both electrodes were filled with the same solution at pH 6.9. The cycle was repeated by drawing again into the first electrode the solution at pH 7.0 (point 3). Reproducible pH differences, precise to + 1 mpH units, were obtained. The apparatus in Fig. 8 can be built to function under aerobic and anaerobic conditions and can be thermostatted. The amount of hemoglobin solution (about 200/zl) that is required for the determination of the difference in pH between deoxy and oxy conditions by this technique using the apparatus in its present asset can be halved by a more convenient design of the loop and of the various connections. In comparison with the combined microelectrode technique, requiring about 60/zl, the stability and the precision of the pH readings were improved significantly.
Conclusions Using the technology for pH measurement described in this article and the buffer capacity of oxyhemoglobin to calculate Bohr protons from 2~pH measurements of hemoglobin solutions under anaerobic and aerobic conditions, about 6-15 mg of hemoglobin for each determination of Bohr protons is required. This could make the study of mutant hemoglobins feasible. Clearly, the buffer capacity of oxyhemoglobin A0 must be the same as that of the mutant. This situation occurs in several cases, e.g., when the mutation replaces a neutral residue with another neutral residue or a neutral residue replaces a group that ionizes in a pH range far from that under investigation. Figure 5B shows that in the pH range 6.5-8.5 the buffer capacity of oxyhemoglobin S, in which two valines replace two glutamic acids on the surface of the protein, is the same within error as that of oxyhemoglobin A0. If the buffer capacity of the mutant in oxyform is significantly different from that of oxyhemoglobin A0, the buffer capacity of the mutant must be measured. To calculate the Bohr protons released from a mixture of two hemoglobins in known proportions and containing the hybrid, one can use the mean value of the buffer capacities of the two hemoglobins. This has been checked using chemically modified hemoglobins, such as hemoglobin reacted with N-ethylmaleimide at the/393 cysteines and hemoglobin car-
[ 12]
PLANCK--BENZINGER RELATIONSHIPS
227
bamoylated at the a-amino groups of the a chains, as substitutes for mutant hemoglobins. 35 Acknowledgments This work was supported by grants from C.N.R. and M.U.R.S.T., Rome.
35 R. Russo, Progress report in fulfillment of a Ph.D thesis.
[12]Application
of Planck-BenzingerRelationships to Biology By PAuLW.
CHUN
Introduction One of the central problems in molecular biology is the formation of the native structure of protein from newly synthesized polypeptide chains. An understanding of protein folding--how proteins achieve complex native forms from the disordered denatured state--is essential in understanding the factors that encode and stabilize particular structural features of proteins and aids in the production of their three-dimensional structures from the amino acid sequence. Under normal physiological conditions, many native protein molecules will adapt the three-dimensional (3-D) structure spontaneously, but a general mechanism to explain the folding transition remains obscure. The principle of spontaneity was formulated on the basis of renaturation experiments in which the chain refolded spontaneously after the ensuing removal of the denaturant agent. 1-3 The behavior of synthetic ribonuclease demonstrated that completed, and not only nascent, chains fold spontaneously under certain experimental conditions, m The fully reduced, random coil polypeptide of ribonuclease can be reoxidized in air to produce the native enzyme, with full enzymatic activity. Consequently, the folding process can be regarded as a transition of the 1 C. B. Anfinsen, E. Huber, M. Sela, and F. M. White, Proc. NatL Acad. Sci. U.S.A. 47, 1309 (1961). 2 C. J. Epstein, R. F. Goldberg, and C. B. Anfinsen, Cold Spring Harbor Symp. Quanr Biol. 27, 439 (1963). 3 C. B. Anfinsen, Science 181, 223 (1973).
METHODS IN ENZYMOLOGY,VOL. 295
Copyright © 1998 by Academic Press All rightsof reproductionin any form reserved. 0076-6879/98 $25.00
ENERGETICS OF BIOLOGICAL MACROMOLECULES
[11]
[ 1 1] S t u d y o f t h e B o h r E f f e c t i n Hemoglobin Intermediates
By
LOUISE BENAZZI, R O S A R I A RUSSO, M A R I L E N A RIPAMONTI, and MICHELE PERRELLA
Introduction Allosteric proteins are devices that transduce signals across a molecule by means of conformational changes. Because allosteric proteins carry out key cellular functions, the mechanisms by which such intramolecular signaling occurs are one of the most intriguing and challenging areas of biophysical research. The tertiary structural changes that underlie the allosteric phenomenon trigger quaternary structural changes in multimeric cooperative proteins. In these complex systems, one type of ligand, the homotropic ligand, binds the subunits cooperatively while the assembly of subunits switches from a low-affinity T to a high-affinity R quaternary conformation. Another type of ligand, the heterotropic ligand, by allosteric binding the subunits modulates the affinity for the homotropic ligand. The thermodynamics of homo- and heterotropic interactions can be a powerful tool for the study of intramolecular signaling provided that the intermediate states of ligation of the protein are accessible to investigation. Hemoglobin is ideally suited for such studies. The intermediate states of ligation with oxygen are not accessible to investigation for technical reasons, but a variety of alternative ligands are available. In landmark studies carried out using the cyanide complex of the ferric subunit, 1-4 extended later to the metal substituted hemoglobins as models of ligation, 5-7 Ackers and collaborators have discovered that the intermediate states of ligation are grouped into three distinct energetic levels. The lowest level corresponds to deoxy hemoglobin, species [01] in Fig. 1. The two monoliganded and one diliganded intermediate, species [11], [12], and [21], form the median level. The remaining species, [22], [23], [24], [31], and [32], belong to the highest level 1 F. R. Smith and G. K. Ackers, Proc. NatL Acad. Sci. U.S.A. 82, 5347 (1985). 2 M. Perrella, L. Benazzi, M. A. Shea, and G. K. Ackers, Biophys. Chem. 35, 97 (1990). 3 M. A. Daugherty, M. A, Shea, J. A. Johnson, V. J. LiCata, G. L. Turner, and G. K. Ackers, Proc. Natl. Acad. Sci. U.S.A. 88, 1110 (1991). 4 M. L. Doyle and G. K. Ackers, Biochemistry 31, 11182 (1992). 5 G. K. Ackers and F. R. Smith, Annu. Rev. Biophys. Chem. 16, 583 (1987). 6 F. R. Smith, D. Gingrich, B. M. Hoffrnan, and G. K. Ackers, Proc. Natl. Acad. Sci. U.S.A. 84, 7089 (1987). 7 p. C. Speros, V. J. LiCata, T. Yonetani, and G. K. Ackers, Biochemistry 30, 7254 (1991).
METHODS IN ENZYMOLOGY, VOL. 295
Copyright © 1998by AcademicPress All rights of reproduction in any form reserved. 0076-6879/98 $25.00
[11]
BOHR EFFECT IN HEMOGLOBIN INTERMEDIATES
209
Ol
(~) (~[~)
11
12
(~'[~)(a[~)
21
(~') (~[3)
22
23
31
24
32
41
(a~.[~.) (a~.[~L) FIG. 1. The 10 ligation states of hemoglobin. The ligand, L, can be 02, CO, NO, or the complex of CN with the heine in the ferric state.
of fully liganded hemoglobin, species [41]. Thus the energies are not distributed into discrete sets corresponding to each ligation state and the two species in which one o~ and one/3 subunit are liganded, i.e., species [21] and [22], are energetically nonequivalent. The inconsistency of this finding with a concerted model of cooperativity s has been the main argument in favor of an alternative mechanism proposed by Ackers, known as the "symmetry rule. ''9 Information on the properties of the intermediates has also been gained by the study of the interaction of hemoglobin with carbon monoxide, both at equilibrium and under dynamic conditions. 1°-12 The large difference in the energetic properties of intermediates [21] and [22] observed in the 8 j. Monod, J. Wyman, and J. P. Changeaux, J. Mol. Biol. 12, 88 (1965). 9 G. K. Ackers, M. L. Doyle, D. M. Myers, and M. A. Daugherty, Science 255, 54 (1992). ,0 M. Perrella, L. Sabbioneda, M. Samaja, and L. Rossi-Bernardi, J. Biol. Chem. 261, 8391 (1986). ~1 M. Perrella, A. Colosimo, L. Benazzi, M. Ripamonti, and L. Rossi-Bernardi, Biophys. Chem. 37, 211 (1990). 12 M. Perrella, N. Davids, and L. Rossi-Bernardi, J. Biol. Chem. 267, 8744 (1992).
210
ENERGETICS OF BIOLOGICAL MACROMOLECULES
[11]
cyanomet model was not confirmed by the CO equilibrium studies. H,~3This apparent discrepancy has been used to refute the general validity of the symmetry rule. 14 This criticism neglects the fact that the mechanism of hemoglobin function, with respect to both ligand affinity and cooperativity, is quantitatively modulated by environmental conditions. The CO model simulates the highly cooperative behavior of oxygen. It apparently conforms to the predictions of the concerted model of cooperativity, as under physiological conditions most of the energy changes in ligand binding that determine cooperativity arise from quaternary state transitions. In contrast, the energetics of the deoxy/cyanomethemoglobin model of ligation indicates that a significant fraction of the energy change in ligand binding arises from tertiary state transitions within the T quaternary state. 9 The study of the association reaction between hemoglobin and CO under nonphysiological conditions, such as high pH, also suggests that the interactions of CO can generate significant cooperativity by means of tertiary structural changes within the T state, is Thus what appears to be a contradiction between ligation models turns out to be a powerful means of investigation into the mechanism, as a wide spectrum of functional properties becomes accessible to measurement as a result of changes in type of ligand or environmental conditions. Bohr Effect of Hemoglobin The binding of oxygen to hemoglobin releases protons. This phenomenon, known as the Bohr effect, occurs with any type of ligand and also on oxidation of the ferrous hemes of the protein. It is due to changes in the pK value of charged groups in response to the conformational changes undergone by the protein in the course of ligand binding. Although the nature of the charged groups is partly a matter of controversy, it is accepted that these groups are located on the a and/3 subunits and that the changes in pK result both from salt bridge and hydrogen bond interactions and from the interactions of certain ions, such as chloride, with charged groups of the protein. 16 Ackers and collaborators have studied the effects on the energetics of the deoxy/cyanomet model of ligation of several heterotropic ligands, including proton, and have shown that the heterotropic ligand does not modify the quaternary state of the protein.t7 Consistent with the energetics 13 M. Perrella and I. Denisov, Methods Enzymol. 259, 468 (1995). 14 S. J. Edelstein, J. Mol. Biol. 257, 737 (1996). 15 C. Ho, Biochemistry 26, 6299 (1987). 16 A. Arnone, R. E. Benesch, and R, Benesch, J. Mol. Biol. 115, 627 (1977). 17 A. Daugherty, M. A. Shea, and G. K. Ackers, Biochemistry 33, 10345 (1994).
[1 1]
BOHR EFFECT IN HEMOGLOBIN INTERMEDIATES
211
is the observation reported by Perrella et al.18that the Bohr protons released by the intermediates in the deoxy/cyanomet system are due to perturbations of the tertiary structures of the subunits within each quaternary structure. Thus the energetics of the intermediate states of ligation in the deoxy/ cyanomet model provides a thermodynamic probe of the tertiary and quaternary structures of the protein, and the Bohr protons released at each ligation state probe the tertiary structures of the liganded subunits in each quaternary state. The information from the two types of thermodynamic studies can provide a means of tracking the pathways of intersubunit communication. Before we describe the methodology for the measurement of the Bohr effect of the intermediates and how this strategy can be potentiated further, the following briefly describes the results of our previous study of the Bohr effects of the deoxy/cyanomethemoglobin intermediates.
Bohr Effects of Hemoglobin Intermediates
Definition of Bohr Effect of Cyanomet Intermediates The protons released on changing a ferrous subunit into a cyanomet subunit cannot be measured directly. Bohr effect of a cyanomet intermediate is described as the difference between the total Bohr effect of hemoglobin at a certain pH value and the protons released by exposing to oxygen the unliganded subunits of the intermediate at the same pH.
Using Deoxy/Cyanomet Model of Ligation The cyanomet model of ligation solves the problem of the mobility of the homotropic ligand that prevents the study of the oxygen intermediates. The high affinity for cyanide of the ferric subunits, as compared with the ferrous subunits, immobilizes cyanide onto these subunits. The rationale for assuming such a complex as a model of ligation is that the crystal structure of cyanomethemoglobin is similar to that of aquomethemoglobin, which is similar to the structure of oxyhemoglobin. However, contrary to aquomethemoglobin, the spin state of the ferric iron of cyanomethemoglobin is low as that of the ferrous ion in oxyhemoglobin.19-22 ~s M. Perrella, L. Benazzi, M. Ripamonti, and L. Rossi-Bernardi, Biochemistry 33, 10358 (1994). 19 M. F. Perutz and F. S. Mathews, J. Mol. Biol. 21, 199 (1966). 20 M. F. Perutz, H. Muirhead, J. M. Cox, L. C. G. Goaman, F. S. Mathews, E. L. McGandy, and L. E. Webb, Nature 219, 29 (1968). 21 M. F. Perutz, H. Muirhead, J. M. Cox, and L. C. G. Goaman, Nature 219, 131 (1968). 22 E. J. Heidner, R. C. Ladner, and M. F. Perutz, J. Mol. Biol. 104, 707 (1976).
zNrSaGZVICSOF BIOLOGICALMACROMOLECULES
212
[1 1]
Only those intermediates that dissociate into identical dimers, such as species [23] and [24], can be studied in a pure form. Intermediates that dissociate into different dimers, indicated in the following as hybrids, such as species [21], [22], [31], and [32], disproportionate rapidly into their parental species, as depicted in Fig. 2. Thus hybrids can be studied under physiological conditions only in the presence of their parental species.
Determination of Bohr Effect of Cyanomet Intermediates The Bohr effects of hemoglobin and of intermediates [23] and [24] are measured using the pure species prepared as in the Experimental Procedures. To determine the Bohr effect of a hybrid species, the protons released by exposing the anaerobic equilibrium mixture of the hybrid and parental species to oxygen are measured. The amount of protons released by the vacant sites of the hybrid species can be calculated if the Bohr effect of the pure parental species and the fraction of each parental species in the mixture are known. The Bohr protons released by the pure hybrid, H+Hyb, are then calculated as follows: H+Hyb = (H+Mix -- H+ParlfParl
-
H+Par2fPar2)(1/fHyb)
(1)
where H+Mix, H+parl, and H+r,ar2 are the protons/tetramer released by the mixture containing parental species and hybrid, and by each of the parental species. The molar fractions of the hybrid and parental species at equilibrium a r e fHyb, fParl, and fPar2" To measure the protons/tetramer, H+Mix, H+Parl, and H+ear2, a known amount of the pure parental species or mixture is deoxygenated, the pH at equilibrium is measured, and the protons released on oxygenation are .... ~
~ . . . . ~'
~ . . . . . ~'
II
[I
If
II
[I
I~. . . .
~
~ ....
~L
6" . . . .
Parent A A
Hybrid AB
II Dimer A
Parent
II ~'
BB
II
aL Dimer B
FIG. 2. Scheme of the dimer exchange reactions between parent species AA (e.g., deoxyhemoglobin) and parent species BB (e.g., cyanomethemoglobin) yielding hybrid species AB and, conversely, of the disproportionation reaction of hybrid AB to yield parents AA and BB. Dashed lines indicate the main intersubunit contacts that break on dimerization.
[1 l]
213
BOHR EFFECT IN HEMOGLOBIN INTERMEDIATES
titrated with NaOH. The molar fractions of the species in equilibrium are determined by cryogenic techniques as described in the Experimental Procedures. The Bohr effect of a parental or hybrid species, B.e.int, is (2)
B.e.int = B.e.Hb -- H+int
where B.e.Hb is the total Bohr effect of hemoglobin and H+int is the amount of protons released on oxygenation of the vacant sites of the intermediates.
Bohr Effects versus pH of Cyanomet Intermediates Three typical curves for the cyanomet intermediates are obtained by measuring the Bohr protons released at different pH values. These pH profiles are shown in Fig. 3 together with the bell-shaped curve of the total Bohr effect of hemoglobin in the same pH range, pH 6.2-9. The difference between the total Bohr effect of hemoglobin and the Bohr effect of a liganded species at the same pH value measures the Bohr protons that are released by the unliganded subunits of that species on oxygenation. Monoliganded Intermediates [11] and [12]. A bell-shaped curve characterizes the pH profiles of the Bohr effect of the monoliganded species [11] and [12] (Fig. 3A), which maintain the T quaternary state at all pH values according to the energetics. 17 Ligation of the c~ or/3 subunit modifies the pK of one or more charged groups, resulting in the release of protons. The pK modification is such that the interactions involved are disrupted at a pH value, in the range of pH 8-8.5, significantly lower than that at which 2.5
B
01
2.0
1,5 ",...
1,0 0.5 0,0 6.5 7 . 0 7 . 5 8.0 8.5
6.5 7.0 7 . 5 8.0 8.5
6.5 7=0 7.5 8.0 8.5
pH
pH
pH
FIG. 3. Bohr effect (B.c.) of intermediates versus pH. (A) Total Bohr effect of Hb, species [01] [also shown in (B) and (C)], and B.e. of species [11] and [12]; (B) B.e. of species [23], [24], [31], and [32]; (C) B.e. of species [21], [22], and calculated sum of species [11] and [12] and B.e. of [21] multiplied by 2. Lines were obtained by fitting experimental data TM using polynomials.
214
ENERGETICS OF BIOLOGICAL MACROMOLECULES
[111
the same groups cease to release protons in hemoglobin (about pH 9.5). The Bohr effect of the unliganded subunits is maintained at pH 8-8.5, in full or in part, despite the possible structural perturbations induced in the protein by ligation of one subunit. Diliganded Intermediates [22], [23], and [24]. These species, which are in the R quaternary state according to the energetics, yield pH profiles of the Bohr effect almost sigmoidal in shape, as in Fig. 3B. At pH 8-8.5, the amount of Bohr protons released by these species equals the total Bohr effect of hemoglobin. This indicates that the liganded and unliganded subunits have the same quaternary and tertiary structures of the subunits of oxyhemoglobin. At neutral and acidic pH, these species release an amount of Bohr protons comparable to that released by monoligation. Under these conditions the amount of protons released by oxygenation of the two unliganded subunits of species [22], [23], and [24] is almost the same as that released by oxygenation of three unliganded subunits in species [11] and [12]. Because species [22], [23], and [24] are in the R quaternary state, 17 pH must induce modifications of the tertiary structures of the unliganded subunits in the R quaternary state, which in releasing Bohr protons mimic the tertiary structures of the subunits in the T quaternary state. Diliganded Intermediate [21]. Ackers and collaborators have found that the change in energy due to cooperativity is the same for the binding of one ligand as for the binding of two ligands yielding the configuration of intermediate [21]. 9 This indicates that, along the pathway in ligand binding centered on intermediate [21], which maintains the T quaternary state, cooperativity arises from changes in tertiary structure of the dimeric halfmolecule. In contrast, according to the concerted model, cooperativity should arise from the increase in the population of molecules in the R quaternary state brought about by the increase in the number of bound ligands. 8 The interpretation of the energetics is entirely consistent with the pH profile of the Bohr effect of species [21] (Fig. 3C). It is not sigmoidal in shape as the profile of the diliganded species [22], which is in the R quaternary state and also liganded at one o~ and one/3 subunit. It is bell shaped as the profile of the monoliganded species, but it is equal neither to the sum of the pH profiles of the two monoliganded species nor to onehalf the total Bohr effect of hemoglobin. This finding is consistent with the energetics, as it indicates that interactions occur in the T quaternary state between the tertiary structures of the subunits of the dimer. Triliganded Species [31] and [32]. The pH profiles of the Bohr effects of these species, which are in the R quaternary state according to the energetics, are also almost sigmoidal in shape as those of the diliganded species [22], [23], and [24] (Fig. 3B) and are interpreted similarly.
[111
BOHR EFFECt IN HEMOGLOBIN INTERMEDIATES
215
Comparison with Oxygen Ligation The Bohr protons released on changing a ferrous into a cyanomet subunit are calculated using the oxygen Bohr effect of the vacant sites. The validity of this procedure is supported by the observation that at pH 7.4 the calculated increments in Bohr effect at each step of the cyanomet ligation along the various pathways in Fig. 1 add up to the total value of the oxygenation Bohr effect of hemoglobin. 18 Furthermore, at pH 7.4 the Bohr effect of the monoliganded cyanomet species, 0.62/0.59 mol H+/ tetramer, is the same, within error, as that (0.64 H+/tetramer) calculated for oxygen under similar conditions.23The overall Bohr effect for the transition from monoligation to the triliganded cyanomet state (0.95-1.4 mol H+/tetramer, depending on the pathway) 18 is not far from the value calculated for oxygen (1.62 _+ 0.27 mol H+/tetramer). 23
Bohr Effect of Mutant Hemoglobins The study of the Bohr effect of the hemoglobin intermediates indicates that a subunit uses the structural changes brought about by ligation to send across the molecules two types of signals. One signal, transmitted to the unliganded subunit across the monomer interface of the dimer, regulates the amount of energy change that occurs within the T quaternary state on binding the second subunit of the dimer. Such a change is slight or negligible in the deoxy/cyanomet model, but is more significant in the CO ligation system. 13The tertiary structural change brought about by diligation in species [21] is not the same as that brought about by monoligation as, as noted earlier, the Bohr effect of species [21] is not equivalent to the sum of the Bohr effect of the two monoliganded species. The other type of signal is sent by the ligated subunit across the dimer interface. Ligation of any one of the subunits of the adjacent dimer promotes the transition from the T to the R quaternary state, as stated by the "symmetry rule." A similar signal is sent across the dimer interface by the diliganded species [21]. Ackers and collaborators have studied the energetics of mutant hemoglobins under oxygenated and deoxygenated conditions to map the intramolecular lines of communication of the subunits interacting with the heme ligand.24,25The combination of the energetics of the intermediates of mutant hemoglobins as a function of pH and of the protons released by the Bohr 23 A. H. Chu, B. W. Turner, and G. K. Ackers, Biochemistry 23, 604 (1984). 24 G. K. Ackers and F. R. Smith, Annu. Rev. Biochem. 54, 597 (1985). 25 V. J. Licata, P. M. Dalessio, and G. K. Ackers, Prot. Struct. Funct. Genet. 17, 279 (1993).
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ENERGETICSOF BIOLOGICALMACROMOLECULES
[1 11
effect of these hemoglobins can be a powerful tool for tracking the pathways of communication across the monomer and dimer interfaces and to clarify the role of the intra- and interchain salt bridges in the quaternary state transition. A serious limitation to such studies is posed by the large amount of protein required by the measurement of the Bohr effect. The following section describes the basic technique used to obtain data summarized earlier and the modifications that could make the study of mutants feasible.
Experimental Procedures
Preparation of Hemoglobin As described later, pH measurements can be carried out on small samples of protein (60-150/~1). However, the precision of the measurements depends on the amount of protons to be determined, which in turn depends on the concentration of the protein sample. Thus methods for the rapid preparation of large quantities of purified hemoglobin and hemoglobin derivatives are a crucial step of the experimental procedure. Hemoglobin A0 is obtained by chromatography on CM-cellulose equilibrated at 4° with 5 mM potassium phosphate, 0.5 mM EDTA, pH 6.8, at 20°. Elution is carried out with 7.5 mM potassium phosphate, 0.5 mM EDTA, pH 7.33, at 20°. Using an 8 × 30-cm bed column, 4 g of hemoglobin from lysed cells yields about 2.8 g of 8 mM (heine concentration) hemoglobin A0 free from minor components as tested by isoelectric focusing (IEF) on a gel plate. The methemoglobin content is about 1.7% according to the cyanomethemoglobin method. 26 The two parental species [23] and [24] in oxyform, i.e., a2+CN-/32 and o~zfl2+cN-, are prepared as follows. Oxyhemoglobin from a blood lysate is oxidized with 1/2 equivalent of ferricyanide for 5 min at 4 ° to yield a mixture of approximately 25% each of HbO2, Hb +, ot2°2f12+, and a2÷/~2%. The mixture is equilibrated by gel filtration with 5 mM potassium phosphate, 0.5 mM EDTA, pH 6.8, at 20° and loaded on a CM-cellulose column equilibrated with the same solvent. Elution is carried out at 4° with 7.5 mM potassium phosphate, 0.5 mM EDTA, pH 7.5, at 20°. Using an 8 × 40-cm bed column loaded with 4 g of hemoglobin and eluted at 300 ml/hr, a complete resolution of species a2°2/~2+ and o~z+/32°2 is achieved in 16-20 hr. The resin portions containing these derivatives are removed by extrusion of the resin bed from the column and suspended in 20 mM Tris-HC1, 50 mM KC1, 1 mM KCN buffer, pH 7.5, at 20° to release the protein. After equilibration in 0.2 M z6K. A. Evelynand H. T. MaUoy,J. Biot Chem. 126, 655 (1938).
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BOHR EFFECT IN HEMOGLOBIN INTERMEDIATES
217
KC1, 1 mM KCN, and concentration, about 0.75 g of 8 mM a2+CN-/~202 and 0.50 g of 8 mM OL202/~2+CN- are obtained. The spectrophotometric analysis of the derivatives yields the following proportions of cyanomethemoglobin: species o/2+CN-~3202, 58.0 q- 1.5 (%); species a2°2/32+cN-, 47.5 +_ 1.0 (%). The use of a 50% oxidized sample of purified hemoglobin A0 does not improve these results, which are similar to those obtained by the alternative, but more laborious, procedure of chain separation and recombination. Because intermediates [23] and [24] are key species for the preparation of all the other intermediates, the procedure for their preparation is crucial for the study of the Bohr effect of the intermediates of mutant hemoglobins. Although our procedure is simple and rapid, it may appear to be wasteful of precious material. However, if the oxy- and methemoglobin fractions, recovered from the column after chromatography, are incubated with trace amounts of ferrocyanide, they reequilibrate with the two 50% oxidized species in a few minutes. In principle, by multiple recycling, all the hemoglobin sample can be used to prepare intermediates [23] and [24].
Preparation of Samples under Anaerobic Conditions The anaerobic equilibration of the intermediates, either pure or in equilibrium with the hybrid species, can be carried out by removing oxygen enzymatically.27 Alternatively, the hemoglobin samples, titrated to a certain pH value, equilibrated, if required, with a concentration of chloride enough to saturate the chloride binding sites and containing an excess of cyanide, are deoxygenated by nitrogen tonometry and placed in glass vials sealed with a silicon cap under a positive nitrogen pressure. The vials are placed in a stoppered cylinder filled with water made oxygen free by the addition of some dithionite. The cylinder is then thermostatted in a water bath. 2 Another procedure, more convenient for the management of a large number of samples, is to connect several capped vials with each other and with a source of humidified nitrogen. The vials are thermostatted in a water bath and a slow stream of nitrogen is maintained during the time of equilibration. 18 The time required by the mixture of parental species to attain equilibrium with the hybrid under anaerobic conditions varies from 24 to 70 hr, depending on the rate of dissociation into dimers of the various tetrameric molecules, which is a function of the state of ligation of the tetramer and of pH. Excess cyanide is needed to make sure that the ferric subunits are saturated by the ligand under all conditions and throughout the equilibra-
27 V. J. LiCata, P. Speros, E. Rovida, and G. K. Ackers,
Biochemistry 29,
9771 (1990).
218
ENERGETICS OF BIOLOGICAL MACROMOLECULES
I111
tion time. A significant loss of cyanide by evaporation in the experiments in which the anaerobicity is maintained by a nitrogen stream could expose the solvent to the ferric subunits facilitating electron transfer reactionsY These reactions are rapid under aerobic conditions and in the presence of ox/redox substances such as ferrocyanide, slow under anaerobic conditions, and negligible, within the time limits of the equilibration experiments, if the met subunits are kept saturated with the ligand. Thus the excess cyanide must be calibrated to the conditions of the experiments. A simple check of the stability of the cyanomet derivatives is to determine the visible spectrum of a sample of cyanomet hemoglobin before and after periods of equilibration under the same conditions of the experiments. As described later, the cryogenic method of analysis of the composition of the mixture of parental and hybrid species provides a "built-in" control of the stability of the cyanomet derivatives at the end of the equilibration. 29
Analysis of Mixtures of Parental Species and Hybrid The composition at equilibrium of the mixture can be calculated if the values of the free energies of dimer assembly into tetramers of the three species are determined using the rate constants of the association and dissociation reactions. The composition of the mixture can be determined directly under the experimental conditions of pH and ionic strength using cryogenic electrophoretic techniques. 3° The aqueous anaerobic mixture of the parent species and their hybrid is injected into a stirred aerobic cryosolvent to attain rapidly a temperature at which the tetramer dissociation reactions are so slow to render the tetramers stable for the time required by the cryogenic separation method. At - 2 5 ° the hybrids of hemoglobins A0 and S are stable for the time, about 20 hr, required to attain the equilibrium in IEF. Hybrids that are unstable under these conditions may be stabilized at lower temperatures. Separation of hemoglobins A0, S, and their hybrid can be carried out at - 4 0 ° by electrophoresis, obtaining resolution of the mixture within 3 hr. 31 Cryogenic IEF and electrophoresis are carried out on tubes of gels made of copolymers of acrylamide and methyl or ethyl acrylate using methylenebisacrylamide as a crosslinker. The higher the proportion of acrylate, the lower the temperature at which the separation can be carried out. Methyl acrylate is the reagent of choice for the lowest temperatures because 28 N. Shibayama, H. Morimoto, and S. Saigo, Biochemistry 36, 4375 (1997). ~9 G. K. Ackers, M. Perrella, J. M. Holt, I. Denisov, and Y. Huang, Biochemistry 36, 10822 (1997). 30 M. Perrella and L. Rossi-Bernardi, Methods Enzymol. 76, 133 (1981). 31 M. Perrella, M. Samaja, and L. Rossi-Bernardi, J. Biol. Chem. 254, 8748 (1979).
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BOHR EFFECT IN HEMOGLOBIN INTERMEDIATES
219
it is more soluble than ethyl acrylate in the methanol-ethylene glycol-water mixtures used as the cryosolvent. Details of the preparation of the gel tubes, procedures for IEF and electrophoresis, and experiments designed to test the equilibration time of the mixtures of parent species and hybrid and to measure the equilibrium composition of the mixture have been published.13.30,32 Cryogenic separations by IEF have the additional advantage that a significant loss of cyanide from the cyanomet subunits can be detected easily, as the fractional charge due to the exposed ferric subunit of a species that has lost its bound cyanide changes its isoelectric point. Partially oxidized carboxyhemoglobin A0 yields nine resolved species by cryofocusing, whereas upon the addition of cyanide to the sample a single component is observed. 32 Thus, cryofocusing provides at the same time a means of resolving the parental and hybrid species and of checking the stability of the cyanomet derivatives during the long incubation time required for the equilibration of the mixture under anaerobic conditions. The quantitative analysis of the protein zones separated by cryogenic IEF or electrophoresis can be carried out by slicing the gels in correspondence to the isolated components, eluting the protein from the gel, and assaying the heme by the pyridine hemochromogen methodfl The total amount of protein sample required by this procedure is about 0.5 mg. Another procedure set up by Ackers and collaborators, requiring even less protein, scans the isolated colored zones of the gel tube using spectrophotometry in the visible range. 27 The cryogenic separation methods can only be used in cases where the parental and hybrid species differ in charge or isoelectric point and the hemoglobins are stable under the conditions of the cryosolvent required by the technique. If the species under study is the hybrid of hemoglobin A0 and a mutant or chemically modified hemoglobin A0 not significantly different in charge from hemoglobin A0, the cryogenic separation of the species can be carried out by replacing hemoglobin A0 with, e.g., hemoglobin S (Glu6/3 ~ Val) or C(Glu6/3 --~ Lys), which differ in charge but are identical to hemoglobin A0 with respect to all other functional properties.
Measurements of pH The measurement of the pH of the samples under anaerobic conditions and the titration with NaOH of the protons released by oxygenation can be carried out using the equipment shown in Fig. 4. Because the pH value of the oxygenated hemoglobin sample is backtitrated to the value of the 32 M. Perrel|a and L. Rossi-Bernardi, Methods Enzymol. 232, 445 (1994).
220
ENERGETICS
OF BIOLOGICAL
MACROMOLECULES
[11]
~ - 6mm ~ [
¢*m~--[
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~4 2 7 F]G. 4. Titration vessel: 1, water-jacketed combined calomel-glass electrode; 2, waterjacketed vessel; 3, vessel cover; 4, nitrogen inlet; 5, oxygen inlet; 6, gas outlet; 7, magnetic bar (adapted from Perrella et al.tS). (Inset) Vessel for pH determination on 60-/.1 samples.
initial a n a e r o b i c c o n d i t i o n s , t i t r a t i o n can b e a v o i d e d if t h e b u f f e r c a p a c i t y o f o x y h e m o g l o b i n as a f u n c t i o n o f p H is k n o w n . T h e b u f f e r c a p a c i t y o f o x y h e m o g l o b i n , B, is d e f i n e d as B = aOH-/ApH[HbO2]
(3)
[ 1 1]
BOHREFFECTIN HEMOGLOBININTERMEDIATES
221
where AOH- are the equivalents of NaOH used for the titration, ApH is the change in pH from the anaerobic to the aerobic conditions, and [HbO2] is the oxyhemoglobin heme concentration. The Bohr protons released by oxygenation of the vacant sites of the intermediates can be calculated as follows: H + (per tetrameric hemoglobin) = 4BApH (4) where 4 is a factor required if the oxyhemoglobin concentration is measured on a heme basis. The apparatus in Fig. 4 was used to titrate with 20 mM NaOH after exposure to oxygen samples of deoxyhemoglobin (1 ml) in the pH range 6.2-9.18 Data on the Bohr effect obtained by this procedure are shown in Fig. 5A. The buffer capacity of oxyhemoglobin calculated from the hpH and zXOH- values recorded in such experiments is shown in Fig. 5B. The Bohr effect of species [11], [23], and [31] calculated from the buffer capacity of oxyhemoglobin and the ApH values measured by exposing to oxygen the pure parental species or their equilibrium mixtures are checked in Fig. 6 against experimental data obtained by the titration method. 18 Clearly, the pH profiles of the Bohr effect of the intermediates can be obtained with the same precision either by titration or by the buffer capacity approach. Such an approach is based on just two pH determinations, under anaerobic and aerobic conditions, and the volume of sample required depends on the technique used for the measurement of pH.
Measurements of p H on Small Samples Combined Electrode Method. The equipment shown in Fig. 4 can be adapted to measure the pH in 60-/xl samples using the device shown in the insert. A glass tube, 4 mm i.d. and 1.5 cm long, is mounted vertically on a Teflon stand, which is steadily rotated inside the thermostatted vessel of Fig. 4 by means of a magnet inserted in the base. The pH of the hemoglobin sample is measured using a combined microelectrode (Ingold INLAG 423, Ag[AgC1, Mettler-Toledo AG, Switzerland). Figure 7 shows the time required to reach a stable pH reading in a solution of deoxyhemoglobin and the pH change observed on oxygenation of this solution using such a device. The values of the Bohr effect of hemoglobin measured by the buffer capacity approach using this method are comparable to those obtained by the titration method (Fig. 5). DifferentialpH Method. Figure 8 shows the scheme of a differential pH m e t e r y which is a development of an earlier apparatus, 34 adapted for 33 Patent applied to U.K. Patent Office by University of Milan on May 2, 1996 (ref. 9609249-9). 34 M. Luzzana, G. Dossi, A. Mosca, A. Granelli, D. Berger, E. Rovida, M. Ripamonti, A. Musetti, and L. Rossi-Bernardi, Clin. Chem. 29, 80 (1983).
222
[11]
ENERGETICS OF BIOLOGICAL MACROMOLECULES
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[11]
BOHR EFFECT IN HEMOGLOBIN INTERMEDIATES
223
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preliminary experiments using concentrated solutions of oxyhemoglobin (10 g/dl) at different pH values. Two capillary glass electrodes (6 tzl inner volume) connected by a loop (90-100/A inner volume) measure the difference in pH between the solutions filling the electrodes. Solutions can be drawn into the system, or driven out, by means of micropumps or, in the modified apparatus shown in Fig. 8, by a gas-tight syringe connected to the outlet of one electrode (electrode B). The other electrode (electrode A) is connected to a reservoir. The total inner volume of the electrodes, loop, and connections to the syringe and reservoir is about 150 ~1. The experi-
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t(min) FI~. 7. Time required to reach the electrode equilibrium before and after oxygenation of the sample (60/xl) of concentrated hemoglobin solution (10 g/dl) contained in the vessel shown in the insert of Fig. 4. (Inset) Precision of the pH readings.
ments were aimed at determining the sensitivity, stability, and reproducibility of the differential pH measurements and the volume of protein solution required to replace completely the solution contained in one capillary electrode with a solution of different pH.
FIG. 8. Scheme of a differential pH meter. It is composed of two glass microelectrodes (A and B) connected by a loop (L). A gas-tight syringe (S) connected to the outlet of electrode B draws the solutions into and out of the electrodes; electrode A is connected to a reservoir (R).
[11]
225
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FIG. 9. Cyclic measurements of the difference in pH between concentrated solutions of oxyhemoglobin (10 g/dl) using the differential pH meter of Fig. 8. Initial conditions: both capillary electrodes are in equilibrium with the same solution at pH 7.0 (point 1). A solution at pH 6.9 is drawn into the system. The initial solution is displaced completely from electrode A by 90-100 /zl of solution at pH 6.9 drawn into the system (point 2). The maximal pH difference between electrode A (pH 6.9) and electrode B (pH 7.0) is recorded at point 2. More solution at pH 6.9 is drawn in to replace the solution at pH 7.0, filling electrode B (point 3). The solution at pH 7.0 is drawn in to replace the solution at pH 6.9, filling electrode A (90-100/zl). A difference in pH of equal magnitude and inverted in sign as shown in point 2 is recorded at point 4.
In the experiment depicted in Fig. 9, the system was filled with a solution of oxyhemoglobin at pH 7.0. A pH difference between the electrodes due to the slightly different signals from the two electrodes in equilibrium with the same solution was zeroed. By acting on the syringe, a solution of oxyhemoglobin of the same concentration at pH 6.9 was then drawn into electrode A (point 1 in Fig. 9) at 10-/zl steps to replace the previous solution at pH 7.0 and at each step the pH was recorded. At the same time, identical amounts of solution at pH 7.0 contained in the loop moved into electrode B. As shown in Fig. 9, the maximal pH difference between the electrodes was measured after drawing in 80-90/~1 of solution at pH 6.9 (point 2). This amount was the minimum volume of solution of oxyhemoglobin at pH 6.9 required to replace the solution at pH 7.0 in electrode A and in
226
E N E R G E T I C S OF B I O L O G I C A L M A C R O M O L E C U L E S
[11]
the tube connecting it to the reservoir under conditions of nonturbulent flow. Because the inner volume of the loop was 90-100/zl, electrode B was still in equilibrium with the initial solution at pH 7.0, which was drawn in from the loop. More solution at pH 6.9 was drawn in to replace the solution at pH 7.0 filling electrode B. The pH difference between the electrodes, inverted in sign, decreased in magnitude until a ApH of _+0.001 was reached again when both electrodes were filled with the same solution at pH 6.9. The cycle was repeated by drawing again into the first electrode the solution at pH 7.0 (point 3). Reproducible pH differences, precise to + 1 mpH units, were obtained. The apparatus in Fig. 8 can be built to function under aerobic and anaerobic conditions and can be thermostatted. The amount of hemoglobin solution (about 200/zl) that is required for the determination of the difference in pH between deoxy and oxy conditions by this technique using the apparatus in its present asset can be halved by a more convenient design of the loop and of the various connections. In comparison with the combined microelectrode technique, requiring about 60/zl, the stability and the precision of the pH readings were improved significantly.
Conclusions Using the technology for pH measurement described in this article and the buffer capacity of oxyhemoglobin to calculate Bohr protons from 2~pH measurements of hemoglobin solutions under anaerobic and aerobic conditions, about 6-15 mg of hemoglobin for each determination of Bohr protons is required. This could make the study of mutant hemoglobins feasible. Clearly, the buffer capacity of oxyhemoglobin A0 must be the same as that of the mutant. This situation occurs in several cases, e.g., when the mutation replaces a neutral residue with another neutral residue or a neutral residue replaces a group that ionizes in a pH range far from that under investigation. Figure 5B shows that in the pH range 6.5-8.5 the buffer capacity of oxyhemoglobin S, in which two valines replace two glutamic acids on the surface of the protein, is the same within error as that of oxyhemoglobin A0. If the buffer capacity of the mutant in oxyform is significantly different from that of oxyhemoglobin A0, the buffer capacity of the mutant must be measured. To calculate the Bohr protons released from a mixture of two hemoglobins in known proportions and containing the hybrid, one can use the mean value of the buffer capacities of the two hemoglobins. This has been checked using chemically modified hemoglobins, such as hemoglobin reacted with N-ethylmaleimide at the/393 cysteines and hemoglobin car-
[ 12]
PLANCK--BENZINGER RELATIONSHIPS
227
bamoylated at the a-amino groups of the a chains, as substitutes for mutant hemoglobins. 35 Acknowledgments This work was supported by grants from C.N.R. and M.U.R.S.T., Rome.
35 R. Russo, Progress report in fulfillment of a Ph.D thesis.
[12]Application
of Planck-BenzingerRelationships to Biology By PAuLW.
CHUN
Introduction One of the central problems in molecular biology is the formation of the native structure of protein from newly synthesized polypeptide chains. An understanding of protein folding--how proteins achieve complex native forms from the disordered denatured state--is essential in understanding the factors that encode and stabilize particular structural features of proteins and aids in the production of their three-dimensional structures from the amino acid sequence. Under normal physiological conditions, many native protein molecules will adapt the three-dimensional (3-D) structure spontaneously, but a general mechanism to explain the folding transition remains obscure. The principle of spontaneity was formulated on the basis of renaturation experiments in which the chain refolded spontaneously after the ensuing removal of the denaturant agent. 1-3 The behavior of synthetic ribonuclease demonstrated that completed, and not only nascent, chains fold spontaneously under certain experimental conditions, m The fully reduced, random coil polypeptide of ribonuclease can be reoxidized in air to produce the native enzyme, with full enzymatic activity. Consequently, the folding process can be regarded as a transition of the 1 C. B. Anfinsen, E. Huber, M. Sela, and F. M. White, Proc. NatL Acad. Sci. U.S.A. 47, 1309 (1961). 2 C. J. Epstein, R. F. Goldberg, and C. B. Anfinsen, Cold Spring Harbor Symp. Quanr Biol. 27, 439 (1963). 3 C. B. Anfinsen, Science 181, 223 (1973).
METHODS IN ENZYMOLOGY,VOL. 295
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