Binding of Inositol Hexakisphosphate to the Oxygenated Derivative of Dromedary (Camelus dromedarius) and Human Hemoglobin: 31P_NMR Study

Paolo Ascenzi, Gino Amiconi, Enrico Rossi, and Anna Laura Segre PA, GA. CNR Centro di Biologia Molecolare, Dipartimento di Scienze Biochimiche, Universita" di Roma "'La Sapienza, "" Roma.--ER. CNR Area della Ricerca, Servizio NMR, Monterotondo Stazione (Roma).--ALS. CNR Area della Ricerca, Istituto di Strutturistica Chimica, Monterotondo Stazione (Roma), Italy

ABSTRACT

Binding of inositol hexakisphosphate (IHP) to the oxygenated derivative of dromedary (Camelus dromedarius) and human hemoglobin (Hb) was investigated by 31P-NMR. The results obtained show that dromedary Hb binds, with different affinity, two HIP molecules per tetramer at distinct sites, while human Hb binds only one HIP molecule per tetramer.

INTRODUCTION

Dromedary (Camelus dromedarius) hemoglobin (Hb) is an ot2/~2 tetramer containing in its sequence [1] all the aminoacid residues considered to be relevant for the functional properties of human Hb [2, 3]. However, at variance with human Hb, which shows only one functionally linked (poly)anion binding site located in a pocket between/~-chains [3, 4], dromedary Hb displays two distinct reactivity-coupled sites with different affinity for (poly)anions, possibly placed between o~- and fl-subunits, respectively [5-8]. Thus, on the basis of dromedary Hb reactivity towards gaseous ligands, it has been .shown that (poly)anion binding to the higher-aff'mity site, equivalent to the functionally linked cleft present in human Hb [5], appears to stabilize the hemoprotein in the T state(s) (according to the Monod et al. terminology [9]),

Address reprint requests to Professor Gino Amiconi, CNR Center for Molecular Biology, Department of Biochemical Sciences, University of Rome "La Sapienza," Piazzale Aldo Moro 5, 1-00185 Rome, Italy.

Journal of Inorganic Biochemistry 35, 247-253 (1989) 24/ © 1989 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/89/$3.50

248 P. Ascenzi et al.

whereas saturation of the binding region with lower affinity for (poly)anions favors the R state(s) of the macromolecule. Many properties of the R state(s), induced in dromedary Hb by the presence of saturating concentrations of @oly)anions, appear to be almost superimposable to those found for the same macromolecule in the absence of (poly)anionic allosteric effecters, suggesting a high similarity in conformation under these two extreme conditions [S-8].

In order to evaluate the interaction of (po1y)anion.s with dromedary Hb in R state(s), inositol hexakisphosphate (IHP) binding to the oxygenated derivative was investigated by 31P-NMR. In addition, in order to shed more light on the structural basis of this process, the interaction of IHP with the oxygenated derivative of human Hb was also studied. The obtained results show that dromedary Hb binds, with different affinity, two IHP molecules per tetramer at distinct sites, while human Hb binds only one IHP molecule per tetramer. As a whole, such findings provide: 1) direct evidence for the existence in dromedary Hb of two distinct (poly)anion binding sites, and 2) information on molecular aspects of IHP interaction with the oxygenated derivative of dromedary and human Hb as well as of differences between the two hemoproteins.

MATERIALS AND METHODS

Oxygenated dromedary and human Hb were prepared from the hemolysate as previously reported [5, lo].

Dromedary and human Hb concentration was determined on the basis of E = 58.4 mM- ’ cm-’ at 577 nm for the tetrameric oxygenated derivative [5, 111.

Both hemoprotein solutions were stripped of any cation and anion (e.g., polyphosphates) by ion exchange chromatography on BioRad Ag-501 X8 [12].

IHP and bis[2-hydroxyethyl]imino-tris-~ydroxymethyl]methane were purchased from Sigma Chemical Co. (St. Louis, USA). 40 (pur. isotop. 99.95% (v/v) D) was obtained from Farmitalia-Carlo Erba S.p. A. (Milano, Italy). The other reagents were from Merck AG (Darmstadt, FRG). All chemicals were of analytical grade and used without further purification.

IHP binding to the oxygenated derivative of dromedary and human Hb was followed by 31P-NMR [13], at pH 7.0 (0.1 M bis[2-hydroxyetbyl]imino-tris-~ydroxymethyl]- methane/HCl buffer system, containing 20% (v/v) 40 plus 0.1 M NaCl) and 25 f 0.2”C.

The l H- 31 P 2D correlation spectroscopy map was obtained as previously reported [14], at pH 5.5 (unbuffered DzO-saturated solution) and 25 f 0.2”C.

31 P-NMR and ‘H-NMR spectra of IHP, in the absence and/or presence of dromedary and human Hb, were recorded on a BRUKER AC 200 spectrometer.

31 P-NMR spectra were run in lo-mm tubes at 81.026 MI-Ix, broad-band decoupled from proton on 8 K size memory; 60’ pulse, a spectral width of 5,000 Hz (corresponding to 1.2 Hz/point digital resolution), and an acquisition time of = 0.8 s were used. The number of scans was 1,200, and a line broadening of 1.0 Hz was applied before the Fourier transformation. All the chemical shift values of 3’ P resonances, given in ppm from external 85 % (v/v) H3PQ, were evaluated by spectral deconvolution [ 151.

Under all the experimental conditions, Hb and IHP concentrations ranged between 2.0 x 1O-5 M and 5.0 x 1O-3 M and 5.0 x 1O-4 M and 2.0 x 1O-3 M, respectively.

Absorption spectra were collected on a VARIAN Cary 219 double-beam spectro- photometer.

HIP BINDING TO Hb: 31P-NMR STUDY 249

2.2 18 14 lx) a6

FIGURE 1. 1H-31 P 2D correlation spectroscopy map of MP in the absence of Hb. Symbols indicate MP resonances of single (A, 0) and double (Cl, 0) intensity. Symbols (A, 0) refer to resonances of single intensity identified and assigned to the unique asymmetric equatorial and

axial 3* Ps of IHP, as detailed on the polyanion tbreedimensional model (external oxygen atoms bound to phosphorus atoms [tilled spheres] were omitted) (modified from Zuiderweg et al. [ 14). i H- and 3’P-NMR spectra were obtained at pH 5.5 (unbuffered DsO-saturated solution), and 25 f 0.2’C. For further details, see text.

REXJLTS AND DISCUSSION

In the absence of Hb, the 31P-NMR spectrum of IHP shows four resonances with an intensity distribution of 1:2:2: 1 (see Figs. 1 and 2) in agreement with the symmetry of the polyanion molecule (see Fig. 1) as observed by x-ray crystallography [16]. No crossing of 31 P resonances of HIP occurs below pH 7.5 [17].

On the basis of the three-dimensional model of IHP [16], the 31P resonances of the polyanion in 40 were identified and/or assigned by the analysis of the 1H-31P 2D correlation spectroscopy map shown in Figure 1. Thus, the 1 H resonance centered at 4.28 ppm, which does not show practically any coupling with its vicinal protons, was assigned to the axial 1 H. From the inspection of Figure 1, it follows that the upfield single intensity 31P signal, geminally coupled to the axial proton of IHP, can be assigned unambiguously to the unique equatorial 31P. Next, the single intensity 31P resonance centered at 2.12 ppm was identified and assigned to the unique asymmetric axial 31P of HIP. Last, the double intensity 31P resonances centered at 0.85 and 1.20 ppm belong to the symmetric axial 31Ps, which, therefore, were identified but not assigned (see Fig. 1).

When HIP is bound to the oxygenated derivative of dromedary and human l-lb, strong variation of the 31P-NMR spectrum can be observed. Figure 2 shows the 31P- NMR spectra of HIP, in the absence and presence of the oxygenated derivative of dromedary and human Hb, as a function of the [Hb]/[IHp] molar ratio, at pH 7.0 and 25’C. The 31P-NMR spectra of HIP with [Hb]/[IHp] = 0 and )l correspond, respectively, to those of the Hb-free and completely Hb-bound states of the polyanion.

In the presence of excess of IHP, 31P resonances of the polyanion vary as a function

250 P. Ascend et al.

3210

Uppm)

FIGURE 2. 3’P-NMR spectra of IHP in the presence of the oxygenated derivative of dromedary and human Hb (left and right panel, respectively) as a function of the [Hb]/[IHP] molar ratio. Symbols used to identify IHP resonances correspond with those shown in Figure 1. 3’P-NMR spectra were obtained at pH 7.0 (0.1 M bis-[2-hydroxyethyl]imino-tris-[hydroxy- methyl]methane/HCl buffer system, containing 20% (v/v) 40 plus 0.1 M NaCl) and 25 f 0.2”C. For further details, see text and Figure 1.

00

3 210

d(ppmI

of the [Hb]/[IHP] molar ratio. Figure 3 shows the chemical shift of the 31P resonances of HIP, in the absence and presence of the oxygenated derivative of dromedary and human Hb, as a function of the [Hb]/[IHP] molar ratio, at pH 7.0 and 25°C. According to Zuiderweg et al. [ 131, the chemical shift values of 31 P resonances of IHP obtained in the presence of slight excess of the oxygenated derivative of dromedary and human Hb are plotted at [Hb]/[IHP] = 1; in fact, the 31P-NMR spectra of IHP at concentration ratios ranging between 1 and 2 are superimposable reflecting the completely I-B-bound state of the polyanion.

All the 31P resonances of IHP shift to lower field proceeding from the I-&free to the completely I-&bound state of the polyanion (see Figs. 2 and 3), a finding that may be taken as indicative of fast exchange of the polyanion between the binding cleft(s) and the solution [ 131. The fast exchange assumption is in agreement with: 1) the number of the 31P resonances of IHP (i.e., four), which is essentially unaffected by changes in the [Hb]/[IHP] molar ratio; 2) previously reported 31P-NMR data on HIP binding to the deoxy and the carbonylated derivatives of human Hb [ 131; and 3) fast kinetics for the reaction of organic polyphosphate(s) with the nitrosylated derivative of dromedary Hb (P. Ascenzi and G. Amiconi, unpublished results) and the deoxy derivative of human

IHP BINDING TO Hb: 31P-NMR STUDY 251

FIGURE 3. Dependence of the chemical shift of IHP in the presence of the oxygenated derivative of dromedary and human Hb (left and right panel, respectively) as a function of the [Hb]l[IHP] molar ratio. (For the sake of clarity, only one out of three measured points has been reported.) The continuous line shown in the left panel follows the 1:2 stoichiometry for IHP binding. Dotted lines shown in left and right panels follow the 1: 1 stoichiometry for MP association. The correlation coefficient of the reportedstraight lines is always higher than 0.99. Symbols used to identify IHP resonances correspond with those shown in Figures 1 and 2. Data were obtained at pH 7.0 and 25 k 0.2”C. For further details, see text and Figures 1 and 2.

Hb [18]. In parallel, the linewidths of all the 3’P resonances, which increase as a function of the [Hb]/[IHP] molar ratio (see Fig. 2), can be accounted for by the weighted average of the widths of the 31P signals of the I&free and the completely I-%-bound polyanion. According to Zuiderweg et al. [ 131, the latter finding means that line-broadening, possibly due to changes in the IHP exchange rates at different [Hb]/ [HIP] molar ratios, is not observed in the systems investigated.

In the presence of the oxygenated derivative of dromedary Hb, the NMR resonance assigned to the unique equatorial 31 P present in IHP shows a double linear behavior as a function of the [Hb]/[MP] molar ratio following a 1:2 stoichiometry; on the other hand, all the other 31P signals shift linearly as the [Hb]/[IHP] molar ratio increases, reflecting a simple 1: 1 binding behavior (see Fig. 3). Such feature differs from that relative to the oxygenated derivative of human Hb, where all the four 31 P resonances of IHP vary linearly increasing the [Hb]/[IHP] molar ratio (see Fig. 3). It is not surprising that the 31P resonance of IHP assigned to the unique equatorial 31P present in the molecule is shifted much more than any other 31P signal upon binding of the polyphosphate to the oxygenated derivative of both dromedary and human Hb, such a resonance being also the most affected one in the polyanion acid-base equilibrium(a) [ 171. These findings are consistent with the higher accessibility of the phosphate group at the equatorial position in the IHP molecule with respect to the axial ones.

As expected from values of the dissociation equilibrium constant(s) for HIP adduct formation with the oxygenated derivative of dromedary Hb (5 x 1 O- 5 M and 1 x 10m4 M [5, 81) and of human Hb (5 x lo-’ M [5, S]), polyanion binding to both hemoproteins follows stoichiometric behavior(s) at the Hb and IHP concentrations employed. I

The inspection of data shown in Figure 3 allows the following relevant considerations: 1) The oxygenated derivative of dromedary Hb binds two IHP molecules per tetramer, with different affinity, at distinct binding sites, as suggested

252 P. Ascenzi et al.

by the abrupt change at [Hb]/@HP] = 0.5 in the chemical shift of the NMR resonance assigned to the unique equatorial 31P of MP. 2) The homogeneous and monotonous dependence of all the four 31P signals of HIP on the whole span of the [Hb]/[MP] molar ratio outlines that the oxygenated derivative of human Hb binds only one polyanion molecule per tetramer, in line with findings previously reported [ 131 for the deoxy and carbonylated derivates at high-ionic strength. 3) The close similarity of the 31P-NMR spectra of the oxygenated derivative of both hemoproteins at [Hb]/[IHP] > 1 (i.e., under conditions in which only the high-affinity binding site of dromedary Hb is saturated with the polyanion) suggests that II-IP binds both hemoproteins at the equivalent sites between P-chains [3, 4, 131. 4) The additional IHP binding site detected in the oxygenated derivative of dromedary Hb might correspond to the aspecific, nonfunctionally linked (poly)anion cleft observable, only at low-ionic strength, in bovine [ 19, 201, horse [21], and human [ 131 Hb, and possibly located between cr-chains [5, 131. 5) When IHP associates with dromedary and human Hb to the specific site(s), a strong shift of the 31P resonance centered at 0.70 ppm in the Hb- free polyanion is observed, suggesting that the equatorial phosphate of HIP preferentially feels the effect of the interaction(s).

As a whole, the data here reported the following: 1) may be taken as a direct and unequivocal evidence for the presence in dromedary Hb of two functionally relevant (poly)anion binding sites, in contrast to human Hb in which only one of the two clefts is specific and reactivity-coupled; and 2) confirm the use of 31 P-NMR spectroscopy as a relevant tool for the investigation of hemoprotein conformation.

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IHP BINDING TO H-b: 31P-NMR STUDY 253

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Received July 20, 1988; accepted October 26, 1988