Cooperative Effect of Inositol Hexakisphosphate, Bezafibrate, and Clofibric Acid on the Spectroscopic Properties of the Nitric Oxide Derivative of Ferrous Human Hemoglobin

Paolo Ascenzi, Albert0 Bertollini, Massimo Coletta, Alessandro Desideri, Bruno Giardina, Francesca Polizio, Roberto Santucci, Roberto Scatena, and Gino Amiconi

PA. Department of Pharmaceutical Chemistty and Technology, University of Turin.--AB, RSa, GA. C.N.R, Center for Mokutar Biology, Department of Biochemical Sciences “Alessandro Rossi Fanelli, ” University of Rome “La Sapienza”.--MC. Depa$ment of Molecular, Cellular and Animal Biology, Univekty of Cametino.-AD. Department of Organic and Biological Chemistty, University of Messina .--BG. C. N. R, Center for Chemistry of Receptors, Institute of Chemistry, Catholic Universig of Rome.--Fp. Depattment of Biology, University of Rome “Tor Vetgata . “--R&L Department of Experimental Medicine

and Biochemical Sciences, University of Rome “Tor Vetgate Italy

ABSTRACT

The cooperative effect of inositol hexakisphosphate (B-W), bezafibrate (BZF), and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of IHP, BZF, and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of 2,3-diphosphoglycerate (2,3- DPG), which have been attributed to a low affinity conformation of the tetramer. Addition to HbNO of two ahosteric effecters together (such as IHP and BZF, or IHP and CFA) further stabilizes the low affinity conformation of the ligated hemoprotein (i.e., HbNO). Moreover, in the presence of saturating amounts of IHP, the affinity of BZF and CFA for HbNO increases by about fifteenfold. Likev&, in the presence of both IHP and BZF, as well as in IHP and CFA, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced with respect to that observed in the

Address reprint, requests and correspondence to: Professor Paolo Ascenzi, C.N.R., Center for MoIecular Biology, Department of Biochemical Sciences “Aksandro Rossi Fanelli,” University of Rome “La Sapienza,” Piazzale Aldo Moro 5,00185 Rome, Italy.

Joumal of Inorganic Biochemis@, So, 263-272 (1993) 263 8 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/93/$6.00

264 P. Ascend et al.

presence of IHP, BZF, or CFA alone, which in turn is lower than that reported in the absence of any ahosteric effector. AII the data were obtained at pH 7.0 (in 1.0 x lo-’ M N-[ZhydroxyethylJ- piperazine-N’-[2-ethanesulfonic acidl/NaOH buffer system plus 1.0 x 10-l M NaCI), as well as at 100 K and/or WC. The results here reported represent clearcut evidence for the cooperative and specific (i.e., functionahy relevant) binding of II-W, BZF, and CPA to Hb.

Abbreviairons

Hb. ferrous human hemoglobin, HbNO, nitric oxide derivative of ferrous human hemoglobin, IHP, inositol hexakisphosphate; BZF, bezafibrate; CFA, clofibric acid; 2,3-DPG, 2,3-diphosphoglycerate.

INTRODUCTION

The low-affinity state of Hb may be induced not only by polyanions (such as IHP and 2,3-DPG) but also by halogenated derivatives of the phenoxy-methylpro- pionic acid (such as BZF and CFA). The latter compounds display antigelling and antihyperlipoproteinemic activities, and bind reversibly to intraerythrocytic Hb, decreasing the oxygen aflkity. From the structural point of view, BZF and CFA interact preferentially with the deoxygenafed derivative of Hb at several binding sites located between one j?-subunit and the two a-chains; on the other hand, IHP and 2,3-DPG associate at the central cavity of the tetramer between the two psubunits. Therefore, since BZF and CFA act cooperatively with IHP and 2,3-DPG when bound to the deoxygenated derivative of Hb, a communica- tion pathway(s) between the clefts where the’ two drugs associate and the polyphosphate binding site must occur. However, it ‘is not unequivocally known whether the cooperativity between BZF or CFA and organic phosphates is still operative in the ligated form of Hb [see Refs. l-111.

Because cooperative ligand binding to oxygen carriers may be described in a simple fashion and can provide some insight for the understanding of more complex systems, the effect of IHP, BZF, and CFA on the spectroscopic properties of HbNO has been investigated at pH 7.0, as well as at 100 K and/or 20°C. In parallel, oxygen aflinity for Hb was determined. The reported results represent clearcut, quantitative evidence for the cooperative and specific (i.e., functionally relevant) binding of allosteric effecters (i.e., IHP, BZF, and CFA) to Hb.

MATERIALS AND METHODS

Oxygenated Hb samples were prepared as previously reported, and stripped of any cation and anion (i.e., allosteric effector) bound to the protein by gel-filtra- tion and ion exchange chromatography 112, 131. Hb concentration was deter- mined on the basis of E = 14.6 mM_’ cm- ’ (at 577 nm), for the oxygenated derivative [12]. The millimolar absorption (mM_’ cm- ‘1 is expressed as l on the millimolar heme basis.

HbNO was obtained, under anaerobic conditions, by sequential addition of sodium dithionite and potassium nitrite (final concentrations 15 mg/ml and 5 mg/ml, respectively) to the oxygenated hemoprotein solutions [6, 71.

IHP, BZF, CFA, and N_[2-hydroxyethyllpiperazine-N’-[2-ethanesulfonic acid] were obtained from Sigma Chemical Co. (St Louis, MO, U.S.). All the other

EFFECT OF IHP, BZF, AND CFA ON HUMAN HBNO 265

products were purchased from Merck AG (Darmstadt, F.R.G.). All chemicals were of analytical grade and used without further purification.

Binding of II-W, BZF (in the absence and presence of 1.0 X 10-l M IHP) and CFA (in the absence and presence of 1.0 X 10-l M IHP) to HbNO was followed by EPR and absorbance spectroscopy. Values of the equilibrium constant for the association of IHP, BZF (in the absence and presence of 1.0 X 10-l M II-IP) and CFA (in the absence and presence of 1.0 X 10-l M IHP) to HbNO (K) were determined at 20°C from absorbance changes accompanying allosteric effector binding [see Refs. 6, 71.

Oxygen binding to Hb was followed by absorption spectroscopy [12]. Values of Pso and n for oxygen association to Hb, in the absence of any allosteric effector, as well as in the presence of IHP, BZF (in the absence and presence of IHP), and CFA (in the absence and presence of IHP) (all 2.0 X 10e2 M), were determined at 20°C from absorbance changes accompanying the dioxygen molecule binding by the tonometric method [12]. The nonsaturating 2.0 x low2 M IHP, BZF, and CFA concentration used is the highest one which does not induce Hb denaturation.

Values of the sedimentation coefficient of the nitrosylated, deoxygenated, and oxygenated derivatives of Hb, in the absence of any allosteric effector, as well as in the presence of IHP, BZF (in the absence and presence of IHP), and CFA (in the absence and presence of IHP) (ranging between 2.0 x 10e2 M and 1.0 X lo- ’ M), were determined at 7°C with a SPINCO model E analytical ultracentrifuge, at 52,000 rev/min; the Hb concentration was 7.5 X 10v5 M [7].

X-band EPR spectra of HbNO, in the absence of any allosteric effector, as well as in the presence of IHP, BZF (in the absence and presence of IHP), and CFA (in the absence and presence of IHP) (all 1.0 x 10-l M), were collected at 100 K on a BRUKER ESP 300 spectrometer [7].

Absorption spectra in the Soret region of the nitrosylated, deoxygenated, and oxygenated derivatives of Hb, in the absence of any allosteric effector, as well as in the presence of IHP, BZF (in the absence and presence of IHP), and CFA (in the absence and presence of IHP) (ranging between 3.0 x lo-’ M and 1.0 x lo- ’ M), were recorded at 20°C on a VARIAN Cary 219 spectrophotometer [7].

All the data were obtained at pH 7.0 in 1.0 X 10-l M N-[Zhydroxyethyl] piperazine-N’-[2-ethanesulfonic acid]/NaOH buffer system plus 1.0 x 10-l M NaCl.

All experiments, in the absence of IHP, BZF, and CFA, as well as in the presence of a single allosteric effector (i.e., IHP, BZF, and CFA) or their mixtures (i.e., IHP and BZF, as well as IHP and CFA), were done on the very same preparation of Hb. Data obtained in the absence of any allosteric effector, as well as in the presence of IHP, BZF, .and CFA are superimposable to those previously reported in the literature [see Refs. 6, 71.

RESULTS AND DISCUSSION

Figure 1 shows the chemical structures of IHP, BZF, and CFA, all acting as allosteric effecters of Hb [see Refs. l-111.

Figures 2 and 3 show the X-band EPR spectra and the absorption spectra in the Soret region of HbNO, in the absence and presence of IHP, BZF, .and CFA (all 1.0 X 10-l M), at pH 7.0 and 100 K or 20°C.

266 P. Ascenii et aL

IHP

0

BZF ~Z-NH-CH~-CH~

y-43

0-C-COOH

&H,

CFA Cl

FIGURE 1. Chemical structures of IHP, BZF, and CFA. In the molecular model of IHP, the external oxygen atoms bound to the phosphorus atoms were omitted for the sake of clarity.

As already known, in the absence of any allosteric effector, the X-band EPR spectrum of HbNO, showing a rhombic shape and a weak hyperfine pattern in the g, region (see Fig. 2, panel A), has been associated to the high affinity state of the macromolecule [see Refs. 1, 2, 71.

Addition of IHP, BZF, and CFA to HbNO induces a transition toward a species characterized by an X-band EPR spectrum with a three line splitting (A, = 1.65 ml3 in the high magnetic field region (g, = 2.01) (see Fig. 2, panels B, C, and D, respectively) [see also Ref. 71. Such a pattern resembles that of HbNO

320 340 320 340

Magnetic Field (mTt

FIGURE 2. X-band EPR spectra of HbNO in the absence of IHP, BZF, and CFA (panel A), as well as in the presence of IHP (1.0 X 10-r M; panel B), BZF (1.0 X 10-l M; panel 0, CFA (1.0 x lo- ’ M; panel D), II-W and BZF (both 1.0 x 10-l M; panel E), and II-IF and CFA (both 1.0 x 10-l M; panel FI. X-band EPR spectra were obtained at pH 7.0, in 1.0 x 10-l M N_[2-hydroxyethyl]piperazine-N’- [Zethanesulphonic acidl/NaOH buffer system plus 1.0 x 10-l M NaCl, and 100 K. The concentration of HbNO was 1.0 X 10e3 M. Setting conditions: 9.42 GHz microwave fre- quency; 20 mW microwave power; 0.10 mT modulation amplitude. For additional experl- mental details, see text.

EFFECT OF IHP, BZF, AND CFA ON HUMAN HBNO 267

A B

100 0 mm _

‘i

t C D

100 -i I

-s 0 (0 ww E F

100 0 IZIQ 400 430 400 430

A Inm)

FIGURE 3. Absorption spectra in the Soret region of HbNO in the absence of II-P, BZF, and CFA (panel A), as well as in the presence of IHP (1.0 x 10-l M; panel B), BZF (1.0 x 10-l M; panel C), CEA (1.0 x 10-l M; panel D), IHP and BZF (both 1.0 x 10-l M; panel E), and IHP and CFA (both 1.0 X 10-l M, panel F). Absorp- tion spectra in the Soret region were obtained at pH 7.0, in 1.0 X 10-l M N_[Zhydroxyeth yllpiperazine-N’_I2-ethanesu.Iphonic acidl/NaOH buffer system plus 1.0 X 10-l M NaCl, and UPC. The concentration of HbNO was 7.5 x 10e5 M. Absorption spectra in the Soret region were recorded in 1 mm path length cuvette. For additional experimental details, see text.

in the presence of 2,3-DPG, which has been attributed to the polyphosphate- induced shift of the conformational equilibrium toward a low affinity state of the ligated hemoprotein (i.e., HbNO) [see Refs. 1, 21.

In the presence of 1.0 X 10-l M IHP, addition of either BZF or CFA to HbNO induces the formation of a species characterized by an X-band EPR spectrum with a more pronounced three line splitting (A, = 1.65 mT) in the high magnetic field region (g, = 2.01) (see Fig. 2, panels E and F, respectively). Such a pattern resembles that observed for horse HbNO, in the presence of IHP, as well as for ruminant and adult loggerhead sea turtle HbNO in the absence of auy allosteric effector, which has been interpreted in terms of a significant stabilization of the low affinity state of nitrosylated hemoproteins [see Refs. 14-161.

In parallel to the IHP-, BZF-, and CFA-induced EPR spectroscopic transition (see Fig. 21, the intensity of the absorbance band in the Soret region of HbNO decreases significantly upon binding of these compounds (see Figs. 3 and 4). In particular, the intensity of the absorbance band in the Soret region of HbNO decreases from 131.0 mM_’ cm-’ at 417.2 nm (in the absence of any allosteric effector) to 101.8 mM_’ cm-‘, 105.2 mM-’ cm-‘, and 107.8 mM_’ cm-’ at 416.2 nm, in the presence of IHP, BZF, and CFA, respectively (all 1.0 x 10-l M) (see Fig. 3, panels A, B, C, and D, respectively) [see also Refs. 1, 71. Moreover, in the presence of saturating amounts of IHP (i.e., 1.0 X 10-l M; see Fig. 4), the addition of either BZF or CFA (both 1.0 x 10-l M) induces a further decrease of the absorbance band in the Soret region of I&NO to 88.2 mM-’ cm-’ and 88.8 mM- ’ cm- ‘, respectively, at 415.3 nm (see Fig. 3, panels E and F, respectively).

The reversible transition brought about by IHP, BZF, and CFA, as well as by 2,3-DPG [see Refs. 1, 2 for reviews] is consistent with either a perturbation of the iron 3d orbital energy, or with the cleavage (or the severe weakening) of the proximal His(F81NE2-Fe bond [see Ref. 21. However, the latter interpretation

268 P. Ascend et al.

FIGURE 4. Ligand binding isotherms for the association of IHP in the absence of BZF and CFA ( v I, BZF in the absence (0) and presence (0) of IHP (1.0 X lo- ’ M), and CFA in the absence (0) and presence (m) of IHP (1.0 x 10-l M) to I-&NO. The concentration of ahosteric effecters (i.e., [L]; MI is that of the free ligand [see Refs. 6,71. The continuous lines were generated from Eq. (1) with the following parameters: K rHP = 1.6( kO.2) x 10e3 M for IHP (in the absence of BZF and CFA); Ku, = 1.5( f 0.2) x lo-* M for BZF (in the absence of II-W); Kc, = 2.8( f 0.3) X lo-* M for CFA (in the absence of IHP); Kam = 9.q f 0.8) x 10e4 M for BZF (in the presence of 1.0 X 10-l M IHP); and Kc, = 2.qf0.2) x 10e3 M for CFA (in the presence of 1.0 x 10-l M IHP) (see Scheme 1). Values of K were obtained with an iterative nonlinear least-squares curve fitting procedure. The data were obtained at pH 7.0, in 1.0 x 10-l M N-[2-hydroxyethyllpiperazine-N’-[2-ethasnesulphonic acidl/NaOH buffer system plus 1.0 x 10-l M NaCl, and 20°C. The concentration of HbNO was 7.5 X 10e5 M. For additional experimental details, see text.

may be preferentially invoked, being supported by Raman and infrared spectro- scopic data of the nitrosylated derivative of ferrous hemoproteins and heme- model compounds [see Refs. 17-191.

Figure 4 shows the ligand binding isotherms for the association of IHP (in the absence of BZF and CFA), BZF (in the absence and presence of 1.0 X 10-l M II-E), and CFA (in the absence and presence of 1.0 x 10-l M IHP) to HbNO at pH 7.0 and 20°C. Applying the minimum model accounting for an apparent single binding site per tetramer, a relation between the IHP, BZF, and CFA equilibrium constant for HbNO (K) and the molar fraction of the ligand-bound nitrosylated hemoprotein (v) may be expressed according to Rq. (1) [see Refs. 6, 71:

y = l/( 1 + K/[L]) (1)

where [L] is the ligand (i.e., IHP, BZF, or CFA) concentration. Equation 1, describing as a function of the allosteric effector concentration the physical quantity Y, has been used to generate the continuous lines shown in Figure 4; the agreement with the experimental data is fully satisfactory (see Fig. 4), giving us confidence that the correct assumptions(s) were made underlying Eq. (1). The simple behavior shown in Figure 4 is also indicative of the absence of HIP-, BZF-, and CFA-induced dimerization of HbNO (even at the highest allosteric

EFFECT OF IHP, BZF, AND CFA ON HUMAN HBNO 269

effector concentration used, 1.0 X 10-r M). This possibility has also been ruled out on the basis of values of the sedimentation coefficient (= 4.1 f 0.1) for HbNO, obtained in the absence and presence of IHP, BZF, and CFA (all 1.0 X 10-l M) [see also Ref. 71Lwhich are indeed typical of a tetrameric state [12]. Next, the simple profile of Y versus lo$L] (see Fig. 4) suggests the absence of significant amounts of denatured HbNO induced by IHP, BZF, and CFA addition (even at the highest concentration used, 1.0 X 10-l M) [see also Ref. 71.

As shown in Figure 4, the affinity of BZF and CFA for HbNO increases by about tifteenfold in the presence of saturating amounts of IHP (i.e., 1.0 X 10-l M). Such a finding indicates that the binding energy of IHP propagates through- out the tetramer, affecting with similar intensity both BZF and CFA association cleft(s).

The low affkity of BZF and CFA for HbNO hinders the determination of the affinity of IHP for the nitrosylated hemoprotein (i.e., the value of Krnr,; see Scheme 1) in the presence of saturating amounts of BZF and CFA (i.e., [BZF] and [CFA] > 1 M; see Fig. 4). However, the value of the equilibrium constant for IHP binding to the binary complexes formed by HbNO and BZF or CFA (i.e., Km,,) can be estimated by the following minimum reaction Scheme 1 [see Ref. 201:

HbNO + IHP

+

X

X:BbNO + IRP

KIHP

F EbNO: IHP

+

X

,- X:AbNO:IHP

where X is BZF or CFA; Kx and Kx, are the equilibrium constants for BZF and CFA binding to HbNO in the absence (Km-r and Kc,) and presence (K BZFt and Kc,,) of IHP; and Km, and KInPI are the equilibrium constants for IHP binding to HbNO in the absence and presence of BZF and CFA, respec- tively. According to Scheme 1, the value of the equilibrium constant for IHP binding to HbNO, in the presence of saturating amounts of BZF and CFA, (Km,,) can be calculated from the experimental values of Krnr, K, (Km.. or K,,), and Kx, (K,,,, or K,rx) (see Fig. 4) applying Eq. (2) [see Ref. 201:

K VIP = K,,,~Wx/K.). (2)

210 P. Ascenn’ et aL

As expected (i.e., K,,l/Ka,, = Kc&Kc,; see Scheme 1, Eq. (11, and Fig. 41, values of Kim,,, calculated according to Eq. (2) from values of K,,, (= l.tif0.2) x 1O-3 M), K,, (= 9LXf0.8) x 1O-4 Ml, K,,, (= lS(f0.2) x lo-* M), KCFW (= 2JXrtO.2) X 10m3 M) and Kc, (= 2.8(f0.3) X lo-* MI, are the same within the error (K,,,, = 9.6(&3) x lo-’ M, and l.l(kO.3) x 1O-4 M, respectively).

From the simple thermodynamic formulation given in Scheme 1, it is possible to evaluate the coupling free energy between IHP and BZF or CFA simultane- ously bound to HbNO (i.e., - RTlnK,,,, + RTlnK,,, = - RTlnKx, + RTlnKx), which turns out to be -6.5 kI mol-‘, at pH 7.0 and 20°C. The negative value of the coupling free energy indicate that the two ligands (i.e., IHP and BZF, or IHP and CFA;) promote each other’s binding, stabilizing the low affinity confor- mation of the tetramer as suggested by EPR and absorbance spectroscopy (see Figs. 2 and 3).

Although the analysis of the data does not allow discrimination between a single ligand binding site per tetramer from multiple independent association pockets, it is clear that IHP, BZF, and CFA associate cooperatively and specifically (i.e., in a functionally relevant way) to HbNO. At variance with what was observed in the case of the deoxygenated derivative of Hb [see Ref. 111, no evidence for multiple classes of binding sites for BZF and CFA has been observed in I-&NO. In the absence of structural data for BZFHbNO and CFAHbNO complexes, a definite conclusion on the location and binding geometry of these functionally relevant allosteric effecters cannot be drawn. Next, the cleft(s) between the Psubunit and the two a-chains suggested for BZF and CFA association to the deoxygenated derivative of Hb [see Refs. 3-5, 9-111 might be assumed as possible candidate(s) even for HbNO. Next, as observed by x-ray crystallography [see Ref. 211, IHP accommodates HbNO in the central cavity of the tetramer between the two pchains.

As reported for IHP and 2,3-DPG binding to Hb [see Refs. 1, 61, values of K for BZF and CFA association to HbNO (K,, = l.!X *0.2) X lo-* M, and K = 2.8(f0.3) x lo-* M; see Fig. 4) are lower than those observed for drug bin”di”np to the two classes of clefts present in the deoxygenated derivative of Hb (BZF: K, = 1.6 x 10e3 M, and K, = 7.7 X 10m3 M; and CFA: K, = 3.4 X 10e3 M, and K, = 1.7 X lo-* M; 5.0 X lo-* M phosphate buffer system plus 1.0 X 10-l M NaCl; pH = 7.4; and 4°C) [see Ref. 111. In turn, in the presence of saturating levels of IHP (i.e., 1.0 X 10-l M; see Fig. 41, the affinity of BZF and CFA for HbNO increases (K,,, = 9.0( f 0.8) X 10m4 M; Kc, = 2.0( f 0.2) X 10m3 M; see Fig. 41, reaching that observed for drug binding to the deoxy- genated derivative of Hb (see above). Such findings underline the role of IHP on switching the tertiary and/or quatemary equilibrium of the ligated hemoprotein (i.e., HbNO) toward a low afhnity conformational state(s) [see Refs. 1, 61. Next, as also previously reported in the case of the deoxygenated derivative of Hb [see Ref. 111, the afhnity of IHP, BZF, and CFA for HbNO can be arranged as follows: IHP > BZF > CFA (see Fig. 4).

As expected, the effect of IHP, BZF, and CFA on the EPR and absorbance spectroscopic properties of HbNO is in keeping with the observed functional behavior for oxygen binding. Thus, in the absence of allosteric effecters, Hb displays an oxygen affinity higher than that shown in the presence of IHP, BZF, or CFA alone; next, after addition of both IHP and BZF, as well as IHP and

TABLE 1.

EFTECl- OF IHP, BZF, AND CFA ON HUMAN HBNO 271

Themmdynamic Parameters (i.e., Values of Pm and n) for Oxygen Binding to Hh in the Absence and in the Presence of IHP, BZF, and CFA at pH 7.0 (in 1.0 x 10-l M NJ2-HydroxyethyllPipemxine-N’_[2-Ethanesulphonic acid]/ NaOH Buffer System plus 1.0 x 10-r M NaCl) and 20°C

Alloateric Effector Pso (nun I-Is> II

None’ 4.3cf0.3) 2.5( f 0.2) II-W 3.0( kO.2) x 10’ 18(*0.1) BZFb 2.a *0.2) x 10’ 1.q f 0.2) CFAb 2.3 f 0.2) x 10’ 1.9(*0.1) IHPb and BZFb 1.2(*0.1)x 102 1.3(*0.1) IHPb and CFAb 1.3(*0.1)x 102 1.3(*0.1)

a The Hb samples were stripped of any anion and cation (i.e., allosteric effecters) according to the literature [12, 131. For additional experimental details, see text. b The allosteric effector concentration was 2.0 X lo-’ M.

CFA, the oxygen affinity for Hb further decreases (see Table 1) [see also Refs. 3-5, 7, 9-111.

As a whole, data here reported allow the following considerations. (i) The association of two allosteric effecters being cooperative, IHP and BZF, or IHP and CFA, are more likely to be bound to the same tetramer than to occupy different molecules of Hb, therefore, the ternary BZF+Ib:IHP and CFA:I-MHP adducts as well as the allosteric effector-free I-Ib predominate over BZF:Hb, CFAHb, and I-Ib:II-IP binary complexes. Such a finding is in line with changes in the value of the Hill coefficient for oxygen binding to I-Ib (i.e., n) which reflect the solution composition (see Table I). (ii) The binary and ternary adducts of I-Ib with IHP, BZF, and CFA display low affinity conformation(s), since a transfer of free energy from the tetramer structure to the surrounding solution occurs. (iii) The close correlation between the spectroscopic, functional and structural properties displayed by I-Ib indeed represents an important confirmation for the use of EPR and absorbance spectroscopy in the elucidation of structure-func- tion relationships in oxygen carrying proteins.

The authors thank Aolfessor Saverio G. ConaZ for helpjid discussions and professor Paola Vecchini for the sedimentation eqetiments. This work was supported by grants from the Italian Ministry for Universi~, Scientific Research, and Technology (Min&-eru per I’Univetsitci e la Ricenza Scientifica e Tecnologica), as well as the Italian National Research Council (Consigiio Nazionale &lle Ricer&e).

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Received July 7, 1992; accepted September 24, 1992