Reinvestigation of the 31P NMR studies of the D myo-inositol 1,2,6 tris(phosphate)-zinc complexes

ELSEVIER

Reinvestigation of the 31p NMR Studies of the D Myo-lnositol 1,2,6 Tris(phosphate)-Zinc Complexes K. Mernissi-Arifi, C. Wehrer, G. Schlewer, and B. Spiess Laboratoire de Pharmacochimie MolJculaire, UPR 421 du CNRS, Facult~ de Pharmacie, 67401 Illkirch Cedex, France

ABSTRACT After reassignment of the phosphorus nuclei for the myo-inositol 1,2,6 tris(phosphate), the structural information drawn about its zinc complexes in a previous study from the 3~p NMR titration curves were reconsidered. It appears, in particular, that in the mononuclear complex, the zinc is mainly coordinated to the equatorial P1 and P6 phosphates, whereas the second cation of the homodinuclear species is stabilized by the axial P2 phosphate.

INTRODUCTION A recent paper which appeared in this journal [1] dealt with the protonation and complexation properties of some myo-inositol tris(phosphates) (Ins(1,2,6)P 3, Ins(1,3,5)P 3, and Ins(2,4,6)P 3) with Cu 2÷, Zn 2÷, Fe 2÷, and Fe 3÷. Potentiometric studies lead to the determination of the nature and the stability of the complexes of the systems previously mentioned. In addition to these studies, the Zn2+-H+-Ins(1,2,6)P3 system was examined by 31p NMR spectroscopy in order to highlight the contribution of each phosphate group on the coordination of the zinc cation. In order to do this, 31p N M R titration curves were performed in the presence and the absence of the metal, and the chemical shifts at a given p H were compared. For reasons that will be further explained, the assignment of the phosphorus resonances were wrongly made, resulting in incorrect conclusions about the coordinating properties of the different phosphates. After

Address reprint requests to: Prof. B. Spiess, FacultY. de Pharmacie, Laboratoire de Pharmacologie Mol6culaire, Universit6 Louis Pasteur, UPR 421 du CNRS, 74 Route du Rhin, PB 24, 67401 Illkirch Cedex, France.

Journal of InorganicBiochemistry, 61, 63-67 (1996) © 1996 Elsevier Science Inc., 655 Avenue of the Americas, NY, NY 10010

0162-0134/96/$15.00 SSD10162-0134(95)00044-O

64 IC Mernissi-Arifi et al.

reestablishing the phosphorus assignment, this paper aims to give the correct interpretation for the 31p NMR and protonation fraction curves, as shown in Figures 3-5 from Ref. [1]. It must be emphasized that all the stability constants, as well as the discussion concerning the overall effect of the zinc coordination on the chemical shift of the phosphorus nuclei, remain valid.

EXPERIMENTAL Resonance peaks of Ins(1,2,6)P 3 were assigned by performing phosphorus-proton 2D correlation experiments at p H 6.50 and 10.50 on a Bruker AC200 Fourier Transform spectrometer. For the 1H-31P chemical shift correlative 2D NMRs, the spectral width in F 1 was 2.2 kHz and in F 2 was 746 Hz. 16 scans for each of the 256 experiments with 1K data points were used. The 256 data points in F 1 were zero filled to 512 W points, p H means the cologarithm of the concentration of H +.

RESULTS AND DISCUSSION T h e 31p NMR titration curves of Ins(1,2,6)P a are shown in Figure 1. It appears

from these curves that the P2 and P6 signals intersect at a high pH. In addition, the p H at which the intersection point can be observed is largely dependent on the concentration of alkali cations present in the medium. For instance, these points arise at p H 9.50 in the absence of potassium and at p H 8.25 in the presence of this cation at a concentration of 0.2 M. This particular behavior of P2 and P6 led, in a first attempt, to the misinterpretation of the phosphorus signals. Thus, subsequently, two different pHs were chosen far enough from that point, i.e., p H 6.50 and p H 10.50, in order to avoid the previous error.

6 6(ppm) O o

v

o o o

P2

o O

0

0086 v

v

V VV o

oo

n

nn

rl

v

o

v

v

V o

o

P6

o

P1

V

O V

O O

V

O

V

O

OV V VOOOOOODO0 0 0

0

n

0 2

3

4

5

6

7

8

9

10

11

12

pH FIGURE 1. Chemical shifts 8 from 3Zp NMR titrations as a function of p H for the H +-[ns(Z,2,6)P3 system.

ZINC COMPLEXES OF INOSITOL LIQUIDS

65

The 1H spectra of Ins(1,2,6)P3 shows the presence of six protons which have been previously assigned [2, 3]. The proton-coupled phosphorus spectrum displays three resonances for the nonequivalent phosphates, split into doublets due to their coupling with the proton of the myo-inositol ring. At p H 6.50, the H1, H2, and H6 protons resonances are 4.13, 4.74, and 4.32 ppm, respectively. The 1H-31P correlative 2D analysis of the contour map shown in Figure 2 indicates that these protons can be associated with the corresponding phosphate resonances at 1.95 ppm (P1), 4.60 ppm (P2), and 3.88 ppm (P6). At p H 10.5 (contour map not shown), the H1, H2, H6 proton resonances at 3.90, 4.60, and 4.15 ppm, respectively, can be correlated with the phosphate resonances at 5.17 ppm (P1), 5.30 ppm (P2), and 5.84 ppm (P6). For the latter pH, it can be seen, as previously stated, that the chemical shifts of P2 and P6 are inverted with respect to those observed at the lower pH. Figure 3 displays the 31p NMR titration curves vs. p H in the presence of zinc. By comparing Figures 2 and 3, it can be seen that the zinc cations largely affect the chemical shifts of the phosphorus nuclei. As demonstrated before [1], this effect is the result of a downfield shift due to the proton displacement and of an upfield shift due to the zinc coordination, the former effect being largely predominant. It is also interesting to note that, with zinc, P1 and P6 show roughly a parallel trend, whereas P2 largely differs. The three phosphates are not equally concerned by the Zn 2÷ binding. P1 experiences the largest effect, indicating its predominant role in the complexation reaction. The difference between the titration curves in the absence and presence of the zinc cations for P2 and P6 is especially important in a p H range

Ilr

5

P1

P6

P2

........ I 4

3

2

Two-dimensional IH-a~P chemical shift correlation contour map showing connectivities via 3JH.P. FIGURE 2.

66

K. Memissi-Arifi et al.

6 8(ppm) v VV V

5

VV V

8 oVooo o ooo []

o ooOa~° °°°°°vo P2

O

O

~

o

P6 v o

o

V

O V

V V

V

O o

o

V 0

0

Oo o

P1

0

0

g[t~ 0

2

7 8 9 10 11 12 pH FIGURE 3. Chemical shifts ~ from 31p NMR titrations as a function of pH for Zn2÷-H + -Ins(1,2,6)P 3 system. 3

4

5

6

going from 4 to 7. Above p H 7, both curves are roughly superimposed for P6 and the complexation curve for P2 is upfield shifted with respect to that of Figure 1. The inspection of the titration and protonation fraction curves (Figure 4) provides some information on the structure of the zinc complexes in solution. Thus, since P1 and P6 show the same "apparent basicity," it is therefore likely that Zn 2÷, in its mononuclear complexes, will be preferentially bound by the equatorial P1 and P6 phosphates. In that case, P2 represents an additional

f. i,p

o

~'g= 980

1.0

O

0.8 0

0.6

8V

Q

O

D

P2 o

0.4

P I ~ v P6 o

O O

V n

0.2

o

O V Q n O O V

~aa~Q V V

0

2

3

4

5

6

7 pH

8

9

10

11

12

FIGURE 4. Protonation fraction fi, p vs. p H for Ins(1,2,6)P 3 in the presence of Zn 2÷.

ZINC COMPLEXES OF INOSITOL LIQUIDS

67

coordination site allowing the formation of a poorly stabilized homodinuclear complex.

REFERENCES 1. K. Mernissi-Arifi, C. Wehrer, G. Schlewer, and B. Spiess, J. Inorg. Biochem. 55, 263 (1994). 2. C. Johansson, J. K6rdel, and T. Drakenberg, Carbohydrate Res. 207, 177 (1990). 3. P. Scholtz, G. Bergmann, and G. W. Mayr, in Methods in Inositide Research, R. F. Irvine, Ed., Raven Press, New York, p. 65. Received February 1L 1995; accepted February 14, 1995