Effect of Inositol Hexakisphosphate on the Spectroscopic Properties of the Nitric Oxide Derivative of Ferrous Horse and Bovine Hemoglobin

Paolo Ascenzi, Massimo Coletta, Alessandro Desideri, Francesca Polizio, Saverio Giovanni Condo’, and Bruno Giardina

PA, MC. C.N.R., Center for Molecular Biology, Department of Biochemical Sciences, University of Rome “La Sapienza”.- AD, FP. Department of Biology, University of Rome “Tor Vergata”.-WC, BG. Department of Experimental Medicine and Biochem- ical Sciences, University of Rome “Tor Vergata, ” Italy

ABSTRACT

The effect of inositol hexakisphosphate (IHP) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous horse and bovine hemoglobin (Hb) has been investigated. In the absence of IHP, the nitric oxide derivative of ferrous horse Hb shows spectroscopic properties similar to those of the corresponding derivative of ferrous human Hb that are generally taken as typical of the high affinity state of tetrametric hemoproteins. Similar to human Hb, the addition of IHP to the nitric oxide derivative of ferrous horse Hb induces a transition toward a species characterized by spectral properties typical of the low affinity state of hemoglobins. Nevertheless, the equilibrium constant for IHP binding to the nitric oxide derivative of ferrous horse Hb (= 1.5 x lo* M-l) is much lower than that reported for the association of the polyphosphate to the same derivative of ferrous human Hb (>3 x lo5 M-l). Conversely, the spectroscopic properties of the nitric oxide derivative of ferrous bovine Hb are characteristic of the low affinity state of tetrameric hemoproteins, both in the absence and in the presence of IHP. These results, taken together with the behavior of the nitric oxide derivative of ferrous human Hb, provide further evidence for the peculiar oxygen binding properties of horse and bovine Hb.

ABBREVIATIONS

Hb, hemoglobin, IHP, inositol hexakisphosphate.

INTRODUCTION

Addition of allosteric effecters (e . g . , IHP) brings about relevant conformational

changes in the nitric oxide derivative of ferrous hemoglobins, which can be detected

Address reprint requests to: Professor Bruno Giardina, Department of Experimental Medicine and Bio- chemical Sciences, University of Rome “Tor Vergata, ” Via Orazio Raimondo 8, 00173 Rome, Italy.

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158 P. Ascenzi et al

by EPR and absorbance spectroscopy. In particular, IHP generally induces in the nitric oxide derivative of ferrous hemoglobins: 1) an EPR transition characterized

mainly by the appearance of a strong hyperfine splitting in the gZ region and 2) an absorbance change characterized by a marked decrease of the extinction coefficient in the Soret region and a slight blue shift of the peak wavelength from 417 nm to 415

nm. Such spectroscopic features, paralleling each other, have been mostly attributed to the perturbation of the His(FQNE2-Fe-NO system geomctq, and have been cor- related to the IHP-induced R 7 T quaternary transition of the nitric oxide derivative

of ferrous hemoglobins 1 i . 21. .A quantitative correlation has been shown between the functional effect of IHP on ligand binding properties cbf hemoproti-ins and the EPR and absorbance spectroscopic transition induced bcs ;liio~ric effe~,~ors ix7 rhl: mtric oxide derivative of ferrous hemoglobins [ 1, 3 -5 j

In order to further characterize such an IHP-induced transition we habe investigated the effect of the polyphosphare on the EPR and absorbance spectroscopic properties of the nitric oxide derivative of ferrous horse and bovine Hh. which display peculiar functional properties. in fact. both hemoproteim show a decrt:ased oxygen affinity with respect to human Hh. this being accompanied by a reduced functional efrecr of polyphosphates, such a; 2.3-diphosphoglycerate and IHP [h-X1

MATERIALS AND METHODS

Horse, human and bovine Hb were prepared as previously reported 19. lo]. and stripped of any cation and anion (e.g.. polyphosphates) bound to the protein by passing the solution through a column of mixed-bed ion exchange resin (Rio Rad AG-501 X8) [l I]. Horce, human. and bovine Hb concentratmn was determined on the basis of E -:~ 15.4 mM ’ cm~ ‘, 13.4 mM ’ cm iI and I4.O mM ’ c-m ’ at 540 nm, respectively, for the carbonylated derivative 19. 101.

The nitric oxide derivative of ferrous horse and bovine Hb was obtained, under anaerobic conditions. hq sequential addition of sodium dithionite and potassium ni- trite (finai concentrations 10 mg/ml and 5 mgiml. reapectivel> 1 rtb the oxygenated hemoprotein solutions [ 1.2 1.

IHP was purchased from Sigma Chemical co. (St. Louis, IMO, USA). All the other products were from Merck AG (Darmstadt, FRG). All chemicals were of analytical grade and used wIthout further purification.

1HP binding to the nitric oxide derivative of ferrous horse Hb was followed by EPR and absorbance spectroscopy 14. 5, 131. The vaiue of the equilibrium constant for the polyphosphate association to the nitric oxide derivative of ferrous horse Hb was determined at 70’ C? frlrn absorbance changes ~~ccompan~i~g H-II: binding [4. 5. 131.

IHP binding to the nitric oxide derivative of ferrous bovine Hb was followed by ” P-NMR spectroscopv [14j. The stoichiometry for the polyphosphate association tc the nitric oxide derivative of ferrous bovine Hb was determined front the shift of the “P resonances of IHP accompanying complex formation [ 141.

Oxygen binding to ferrous horse, human, and bovine Hb was followed by ab- sorbance spectroscopy [9J. Values of P 50 for oxygen association to ferrous horse. human, and bovine Hb. in the absence and presence 01’ saturating IHP concentration ([IHP] > 1 .O x I II I’ M), were determined from absorbance changes accompanying the dioxygen molecule binding, by the tonometric method [9].

All the data were obtained at pH 7.0, in 0.1 IM N-[2-hydroxyethyl]piperazine-N’- [2-ethanesulphonic acid]/ NaOH chloride-free buffer system,

SPECTROSCOPIC PROPERTIES OF HORSE AND BOVINE HbNO 159

I I 0- 310 330 350 400 440

Magnetic Field (mTI i. (nml

FIGURE 1. X-band EPR spectra (panels A and B) and absorbance spectra, in the Soret region, (panels A ’ and B ‘) of the nitric oxide derivative of ferrous horse (panels A and A ‘) and bovine (panels B and B’) Hb, in the absence (spectra a) and presence (spectra b) of 0.1 M IHP. Spectra were obtained at pH 7.0 in 0.1 M N-[2-hydroxyethyl]piperazine-N’- [2-ethanesulfonic acid]/NaOH chloride-free buffer system. For the sake of clarity, the absorbance spectrum in the Soret region, of the nitric oxide derivative of ferrous bovine Hb in the presence of 0.1 M IHP (see panel B’, spectrum b) was increased by E = 10.0 mM- ’ cm-‘. X-band EPR spectra and ab- sorbance spectra in the Soret region were recorded at 100 K and 20°C respectively. The concentration of the nitric oxide deriva- tive of ferrous horse and bovine Hb ranged between 7.5 x 10m5 M and 1.0 x 10m3 M. Setting conditions for X-band EPR spectra: 9.42 GHz microwave frequency; 20 mW microwave power; 0.10 mT modulation am- plitude. Absorbance spectra in the Soret re- gion were recorded in 1 mm path length cuvettes. For additional experimental details, see text.

EPR spectra of the nitric oxide derivative of ferrous horse and bovine Hb, in the absence and presence of IHP, were collected at 100 K on a BRUKER ESP 300 spectrometer.

Absorption spectra of the deoxygenated, carbonylated, nitrosylated and/or oxy- genated ferrous horse, human, and bovine Hb, in the absence and presence of IHP, were recorded at 20°C on a VARIAN Cary 219 spectrophotometer. The millimolar absorption (mM_i cm-i) is expressed as e on the millimolar heme basis.

31P-NMR spectra of IHP, in the absence and presence of the nitric oxide derivative of ferrous bovine Hb were recorded at 20°C on a BRUKER AC 200 spectrometer.

RESULTS AND DISCUSSION

Figure 1 shows the X-band EPR spectra and the absorption spectra, in the Soret region, of the nitric oxide derivative of ferrous horse and bovine Hb at pH 7.0, in the absence and.in the presence of IHP (0.1 M).

The X-band EPR spectrum of the IHP-free nitric oxide derivative of ferrous horse Hb (Fig. 1, panel A, spectrum a), shows a rhombic shape and a weak hyperfine pattern in the g, region. Such a spectrum is very similar to that described for the cor- responding derivative of human Hb under the same conditions, and can be associated to the high affinity state of the macromolecule (see Refs. 1, 2 for reviews).

Addition of IHP to the nitric oxide derivative of ferrous horse Hb induces a tran- sition toward a species characterized by an X-band EPR spectrum with a three line splitting (A, = 1.65 mT) in the high magnetic field region (g, = 2.01) (Fig. 1, panel A, spectrum b). Such a pattern resembles that of the nitric oxide derivative of

160 P. Ascenri et ul

SPECTROSCOPIC PROPERTIES OF HORSE AND BOVINE HbNO 161

presence of IHP. These features suggest that the nitric oxide derivative of bovine Hb, similarly to that reported for the same derivative of opossum Hb [3, 241, is already in a low affinity conformation, even in the absence of IHP. Indeed, IHP binding to the nitric oxide derivative of ferrous bovine Hb was established by 31P-NMR. All the 31P resonances of IHP shift linearly to lower field as the [Hb]/[IHP] molar ratio increases, reflecting a simple 1: 1 binding behavior.

The EPR and absorbance spectroscopic properties of the nitric oxide derivative of ferrous bovine Hb are consistent with the observed functional behavior for oxy- gen binding. Thus, the intrinsically reduced oxygen affinity of bovine Hb, even in the absence of IHP (Table l), which has been attributed to amino acid substitutions of the N-terminal region of P-chains (i.e., the deletion of ValNAl and the substi- tution of HisNA2 by MetNAl) [6], is indeed accompanied by a reduced effect of poiyphosphates on ligand binding properties (Table 1).

Beside the g, region analyzed thus far, the X-band EPR spectra of the nitric oxide derivative of ferrous horse Hb, in the presence of IHP, and of ferrous bovine Hb, both in the absence and in the presence of the polyphosphate, display some differences in the gx region. Thus, the hyperfine splitting in the low magnetic field region (&; = 2.09) of the X-band EPR spectrum of the nitric oxide derivative of ferrous bovine Hb (Fig. 1, panel B, spectra a and b) is similar to that of the corresponding derivative of human Hb, under the same conditions 1241, but differs significantly from that of the nitric oxide derivative of ferrous horse Hb (Fig. 1, panel A, spectrum b). As suggested by John and Waterman [24], such a finding may reflect different stabilization modes of the bound nitric oxide molecule by hydrogen bonding with the NE2 atom of the distal His E7 residue. However, in the absence of comparable structural information no further conclusion can be drawn on the origin of this spectroscopic difference.

The close correlation between the functional and spectroscopic properties displayed

by horse, human, and bovine Hb indeed represents an important confirmation for the use of EPR and absorbance spectroscopy in the elucidation of the mechanisms underlying ligand binding in hemoproteins.

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Received December 19, iY89; accepted Februrrry 28, 1990