Effect of Inositol Hexakisphosphate on the Spectroscopic Properties, of the Nitric Oxide Derivative of Ferrous Naturally Glycated Human Hemoglobin HbA1,

Paolo Ascenzi, Alessandro Desideri, Gino Amiconi, Albert0 Bertollini, Martin0 Bolognesi, Massimo Castagnola, Massimo Coletta, and Maurizio Brunori

PA, GA, AB, MCo, MBr. C.N.R., Center for Molecular Biology, Department of Biochemical Sciences, University of Rome ‘La Sapienza”.-AD. Department of Biology, University of Rome “Tar Vergata”. -MBo. Department of Genetics and Microbiology, Section of Crystallography, University of Pavia .-MCa. Institute of Chemistry, Catholic University of Rome “Sacro Cuore, ” Italy

ABSTRACT

The effect of inositol hexakisphosphate (IHP) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous naturally glycated human hemoglobin HbA,, (HbA,,NO) has been investigated quantitatively. The results obtained show that 1) both in the absence and presence of IHP, the EPR and absorbance spectra of HbA,,NO show the same basic characteristics described for the nitrosyl derivative of ferrous Hb&, the nonglycated major component of human hemoglobin (Hb&NO); and 2) HbA!,NO binds IHP with an apparent dissociation equilibrium constant (19 = 1.8 x 10e2 M), which is at least four orders of magnitude higher than that estimated for the polyphosphate interaction with I-R&NO (6 3 x 10m6 M). These data provide further independent evidence that interaction(s) of polyphosphates at the specific cleft between P-chains along the dyad-axis is sterically hindered in HbA,, by the presence of the two glucose residues covalently bound to the N-termini of P-chains, this finding being in agreement with the reduced effect of polyanions on HbA1, spectral and ligand-binding properties.

K&WORDS

Ferrous Human HbA,,NO; Spectroscopic (EPR and absorbance) properties; IHP, effect of, on spectroscopic properties of human HbA,,NO

Address reprint requests to: Professor Maurizio Brunori, Department of Biochemical Sciences, University of Rome “La Sapienza,” Piazzale Aldo Moro 5, 00185 Rome, Italy.

Journal of Inorganic Biochemistry 34, 19-24 (1988) 19

0 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017 0162-0134/88/$3.50

20 P. Ascenzi et al.

INTRODUCTION

Human red blood cell hemolysate contains, in addition to the major component Hb&, several glycated minor hemoglobins [I]. Chemical analysis of HbA,,, the most abundant fraction of such glycated components, has shown that the a-amino group of the valyl residue at the N-terminus of the two P-chains forms a stable ketoamine linkage with glucose through the Schiff base labile intermediate and the Amadori rearrangement [2].

Functional studies (see Refs. 3-7) indicate that, by comparison with HbAa, in HbAi, 1) the overall affinity is slightly higher; 2) cooperativity in oxygen binding is reduced; 3) the lower part of the oxygen binding isotherm is markedly different (the lower asymphote extending up to P = 0.4); and 4) the effect of polyanions on spectral and ligand binding properties is decreased. The latter finding has been related to the unfavorable interaction(s) of polyanions with residues in the cleft between P-chains along the dyad-axis, because of steric hindrance by the two glucose residues covalently bound to the N-termini of the two P-chains (see Ref. 8).

In order to further explore polyphosphate binding to HbA,,, the effect of inositol hexakisphosphate (IHP) on the spectroscopic (EPR and absorbance) properties of the nitric oxide ferrous derivative (HbAi,NO) has been investigated quantitatively, and analyzed in parallel with that reported for the nitrosyl derivative of ferrous Hb& (HbA,$JO) (see Refs. 9, 10 for reviews).

The results indicate that 1) IHP binding to HbA,,NO induces EPR and absorbance changes identical to those reported for Hb&NO; and 2) the affinity of IHP for HbA,,NO is at least four orders of magnitude lower than that observed for Hb&NO.

MATERIALS AND METHODS

HbAt, was purified by preparative cation exchange chromatography on BioRex 70 [l 11. HbAt, preparations always showed a level of purification greater than 95 % , as checked by isoelectric focusing of whole-native hemoglobin and its denatured globin chains [ 111. Moreover, the specificity of the alteration at the o-amino groups of the /3- chains’ N-termini has been checked by RP-HPLC of the product(s) of tryptic digestion

WI. HbAi, concentration was determined on the basis of E = 14.6 x lo3 M- * cm-’ at

577 nm for the oxygenated derivative [ 131. The hemoprotein solution was stripped of any cation and anion (e.g., polyphos-

phates) by mixed ion exchange chromatography on BioRad AG-501 X8 [ 141. HbA,,NO was obtained, under anaerobic conditions, by sequential addition of

sodium dithionite and potassium nitrite (final concentrations 10 and 5 mg/rnl, respectively) to the oxygenated hemoprotein solutions [ 151.

IHP was purchased from Sigma Chemical Co. (St. Louis, USA). The other reagents were from Merck AG (Darmstadt, FRG). All chemicals were of analytical grade and used without further purification.

IHP binding to HbA,,NO was followed spectroscopically at pH 7.0 (0.1M bis[2- hydroxyethyl]imino-tris-[hydroxymethyl]methane/HCl buffer system plus 0.1 M NaCl) [6, 10, 16-191.

X-band EPR spectra were collected at 110 K on a Varian E-9 spectrometer. Absorption spectra in the Soret region were recorded at 20°C on a Varian Cary 219

spectrophotometer. The molar absorption (M-’ cm-‘) is expressed as E on the molar heme basis.

PROPERTIES OF HUMAN HbA,,NO 21

The pH levels of the solutions were determined before and after each experiment at 20°C with a Radiometer model 5 1 pH meter.

RESULTS AND DISCUSSION

Figure 1 shows the EPR and absorption spectra of HbA,,NO, in the absence and presence of IHP (0.1 M), at pH 7.0. Such spectra show the same basic characteristics described for HbA,,NO, in the absence and presence of allosteric effecters (see Refs. 9, 10 for reviews).

The EPR spectrum of the polyphosphate-free I-IbA,,NO (Fig. l), showing a rhombic shape and a weak hyperfine pattern in the g, region, has been associated to the high-affinity state of the macromolecule [6, 9, 10, 16, 17, 20-251. Addition of 0.1 M IHP to the system brings about a transition towards a species characterized by an EPR spectrum with a three-line splitting (A, = 1.7 mT) in the high magnetic field region (g, = 2.01) (Fig. 1); such a pattern has been attributed to the low-affinity state of the hemoprotein [6, 9, 10, 16, 17, 20-251. The transition induced by IHP is not a priori inconsistent with either a perturbation of the iron 3d orbital energy [lo, 23, 241, or with the cleavage (or the severe weakening) of the proximal His(F8)-Fe bond [9, 10, 17, 2 1, 22, 26, 271. However, the latter interpretation is preferentially supported by Raman and infrared spectroscopic data of nitrosyl hemoproteins and heme-model compounds [21, 26, 281, as well as by theoretical calculations [29].

As shown in Figure 1, the intensity of the absorbance band in the Soret region of

FIGURE 1. X-band EPR (left panel) and absorbance (right panel) spectra of HbA,,NO in the absence (A) and presence (I3) of 0.1 M IHP. Spectra were obtained at pH 7.0, 0.1 M bis[2- hydroxyethyl]-imino-tris-[hydroxymethyl]methane/HCl buffer system plus 0.1 M NaCl. X- band EPR and absorbance spectra were recorded at 110 K and 2O”C, respectively, HbAi,NO ranged between 7.5 x loss M and 2.0 x 10e4 M. Setting conditions for X-band EPR spectra: 9.15 GHz microwave frequency; 20 mW microwave power; 0.12 mT modulation amplitude. Absorbance spectra were recorded in 2 mm path length cuvete. For additional experimental details, see text.

J 1 c

Magnetic Field (mT) Wavelength (nm)

325 335

100

50

22 P. Ascenzi et al.

log BHP] (M)

FIGURE 2. Binding of IHP to HbA,,NO, as monitored by X-band EPR (squares) and absorbance (circles) spectroscopy. The concentration of IHP is that of the free polyphosphate [19]. The continuous line represents the best theoretical titration curve calculated according to Eq. 1 with K = 1.8 x 10e2 M. The data were obtained at pH 7.0 and 110 K (for X-band EPR spectra) and 20°C (for absorbance spectra). For additional experimental details, see Figure 1 and text.

HbAI,NO decreases significantly upon IHP binding. Such absorption spectral change (AE = 30 mM - 1 cm ’ at X = 416.5 nm) is identical to those observed for proton and/ or (poly)anion binding to the nitric oxide derivative of ferrous monomeric and tetrameric hemoproteins [9, 17-191, and parallels the IHP-dependent transition monitored by EPR spectroscopy (Fig. 2), in both cases reflecting the perturbation of the His(F8)-Fe-NO system geometry [9, 17-191. As previously reported [18, 191, this finding suggests that the EPR measurements, although performed at 110 K, correspond to the equilibrium condition(s) prevailing at 2O”C, where absorbance spectra were recorded.

IHP binding to HbA,,NO has been measured by EPR and absorbance spectroscopy at different concentrations (Fig. 2). Applying a simple model accounting for a single binding site per tetramer, the molar fraction (P) of the system, as a function of IHP concentration, may be expressed as follows [19]:

P= 1 * {l/(1 + [IHPJIK)} (1)

where K is the apparent dissociation equilibrium constant for the polysphosphate. Eq. (1) has been used to generate the continuous line shown in Figure 2 with the value of K = 1.8 x 1 Oe2 M. In spite of the uncertainty in the evaluation of the asymptote at high polyphosphate concentration, the agreement with the experimental data is fully satisfactory (Fig. 2) giving us confidence on the correct assumption underlying Eq. (1). This simple behavior is indicative of the absence of IHP-induced dimerization in HbA,,NO (even at the highest concentration, 0.1 M). In order to prevent such a possibility, the concentration of HbA,,NO used in these experiments (ranging between 7.5 x 10ms M and 2.0 x lo- 4 M) exceeded by far the value of the tetramer-dimer

dissociation equilibrium constant of ferrous ligated (i.e., carbonylated) hemoglobin at pH = 7.0, in the presence of 0.1 M IHP (= 2.2 x 10e6 M) [30].

The value of K for IHP binding to HbA,,NO (= 1.8 x 10e2 M) is at least four

PROPERTIES OF HUMAN HbA,,NO 23

orders of magnitude higher than that estimated for the interaction with the Hb&NO (<3 x 10T6 M; [17]). Following this, because of the presence of the two glucose molecules into the polyphosphate specific cleft between P-chains along the dyad-axis pocket, IHP binding to I-IbA,,NO is unfavorable by at least 5 kcal/mol.

The lower affinity of IHP for HbAi,NO with respect to I-R&NO accounts for the negligible effect of polyphosphates on the spectral and ligand binding properties of HbAr,, at least up to 1 x 10d3 M IHP [3-71. On the other hand, in Hb&, the effect of IHP on spectral and ligand binding properties is essentially complete already at 1 x

10 - 3 M concentration [3 1, 321, as expected from the high affinity of the polyphosphate for HbAsNO [17].

The dramatically decreased affinity of IHP for I-IbA,,NO reflects the unfavorable interaction(s) of the polyphosphate at the specific cleft between P-chains along the dyad-axis [3, 5, 8, 331. Such a finding could arise from two contributions: 1) the loss of the two positive charges at the N-termini of the P-chains, which are chemically blocked, and 2) the steric hindrance exerted by the two covalently bound glucose molecules. The first contribution plays certainly some role, as demonstrated by the reduced effect of IHP on the oxygen binding properties of human hemoglobin Raleigh, where the N-terminus of P-chains is an acetylalanyl residue [34]; however, the second contribution may also be important, at least to the same extent as the first. Thus, the effect of IHP on oxygen binding properties of HbA,, [7], where steric and electrostatic effects are both present, is very much reduced not only with respect to Hb& [31, 321, but also with respect to human hemoglobin Raleigh, where only the electrostatic contribution is operative [34]. Moreover, it is relevant that from molecular modeling of HbAt,, no clear-cut orientation of the two glucose molecules bound to the P-chains could be detected, implying no stable occupancy of the cavity along the dyad-axis.

As a whole, the data here reported are of particular relevance 1) representing the first quantitative estimate of the reduced effect of polyphosphates on HbA,, spectral and ligand binding properties, and 2) possibly being of general significance applying not only to HbA,,, but also to hemoglobins naturally and synthetically reacted at the N-termini of P-chains [5-8, 34, 351.

This work was supported by grants of the Italian Ministry of Education (Ministero della Pubblica Istruzione) and the Italian National Research Council (Consiglio Nazionale delle Ricerche).

REFERENCES

1. D. W. Allen, W. A. Schroeder, and J. Balog, J. Am. Chem. Sot. 80, 1628 (1958). 2. R. J. Koenig, S. H. Blobstein, and A. Cerami, .I. Biof. Chem. 252, 2992 (1977). 3. H. F. Bunn, D. N. Haney, K. N. Gabbay, and P. M. Gallop, Biochem. Biophys. Res.

Commun. 67, 103 (1975). R. Fluckiger and K. H. Winterhalter, in Biochemical and Clinical Aspects of Hemo- globin Abnormalities, W. S. Caughey, Ed., Academic Press Inc., New York, 1978, pp. 205-214. M. McDonald, M. Bleichman, H. F. Bunn, and R. W. Noble, J. Biol. Chem. 254, 702 (1979). K. Nagai, Y. Enoki, A. Kareko, and H. Hori, Biochim. Biophys. Acta 623, 376 (1980).

24 P. Ascenzi et al.

7. M. Coletta, G. Amiconi, A. Bellelli, A. Bertollini, J. C&sky, M. Castagnola, S. Condo, and M. Brunori, J. Mol. Biol., in press (1988).

8. S.-H. Chiou, L. M. Garrick, and M. L. McDonald, Biochemistry 21, 13 (1982). 9. M. F. Peru& Annu. Rev. Biochem. 48, 327 (1979).

10. W. E. Blumberg, Methods Enzymoi. 76, 312 (1981). 11. M. Castagnola, P. Caradonna, L. Cassiano, C. Degen, F. Lorenzin, D. Rossetti, and

M. L. Salvi, J. Chromatog. 307, 91 (1984). 12. W. A. Schroeder, J. B. Shelton, J. R. Shelton, and D. Lowars, J. Chromatogr. 174,

385 (1979). 13. E. Antonini, and M. Brunori, in Hemoglobin and Myoglobin in their Reactions with

Ligands, A. Newberger and E. L. Tatum, Eds., North-Holland, Amsterdam, 1971. 14. G. Amiconi, and B. Giardina, Methods Enqymol. 76, 533 (1981). 15. T. Yonetani, H. Yamamoto, J. E. Erman, J. S. Leigh, Jr., and G. H. Reed, J. Biol.

Chem. 247, 2447 (1972). 16. H. Rein, 0. Ristau, and W. Scheler, FEBS Lett. 24, 24 (1972). 17. M. F. Perutz, J. V. Kilmartin, K. Nagai, A. Szabo, and S. R. Simon, Biochemistry

15, 378 (1976). 18. P. Ascenzi, G. M. Giacometti, E. Antonini, G. Rotilio, and M. Brunori, J. Biol.

Chem. 256, 5383 (1981). 19. P. Ascenzi, R. Santucci, A. Desideri, and G. Amiconi, J. Znorg. Biochem., 32, 225

(1988). 20. E. Trittelvitz, H. Sick, and K. Gersonde, Eur. J. Biochem. 31, 578 (1972). 21. J. C. Maxwell and W. S. Caughey, Biochemistry 15, 38 (1976). 22. A. Szabo and M. F. Peru@ Biochemistry 15, 4427 (1976). 23. M. Chevion, W. E. Blumberg, and J. Peisach, in MetaI-Ligand Interactions in Or-

ganic Chemistry and Biochemistry, Part 2, B. Pullman and N. Goldblum, Eds., D. Reidel, Dordrecht, 1977, pp. 153-162.

24. M. Chevion, A. Stem, J. Peisach, W. E. Blumberg, and S. R. Simon, Biochemistry 17, 1745 (1978).

25. M. E. John and M. R. Waterman, FEBS Lett. 106, 219 (1979). 26. B. B. Wayland and L. W. Olson, J. Am. Chem. Sot. 96, 6037 (1974). 27. H. Kon, Biochim. Biophys. Acta 379, 103 (1975). 28. K. Nagai, C. Welbom, D. Dolphin, and T. Kitagawa, Biochemistry 19, 4755 (1980). 29. S. K. Mun, J. C. Chang, and T. P. Das, Proc. Natl. Acad. Sci. USA 76, 4842

(1979). 30. R. D. Gray, J. Bioi. Chem. 255, 1812 (1980). 3 1. K. Imai and T. Yonetani, J. Biol. Chem. 250, 7093 (1975). 32. R. Edalji, R. E. Benesch, and R. Benesch, J. Biol. Chem. 25, 7720 (1976). 33. G. Fermi and M. F. Perutz, in Atlas of Molecular Structures in Biology. 2. Haemo-

globin and Myoglobin, D. C. Phillips and F. M. Richards, Eds., Clarendon Press,

Oxford, 1981. 34. W. F. Moo-Penn, K. C. Bechtel, R. M. Schmidt, M. H. Johnson, D. L. Jue, D. E.

Schmidt, Jr., W. M. Dunlap, S. J. Opella, J. Bonaventura, and C. Bonaventura, Bio- chemistry 16, 4872 (1977).

35. A. Bellelli, R. Ippoliti, D. Currell, S. G. Condo, B. Giardina, and M. Brunori, Eur. J. Biochem. 161, 329 (1986).

Received May 5, 1988; accepted May 6, 1988.