COMPLETE RESOLUTION OF THE MICROSCOPIC - PROTONATION EQUILIBRIA OF D-MYO-INOSITOL 1,2,6-TRIS(PHOSPHATE) AND RELATED-COMPOUNDS BY P-31 NMR AND POTENTIOMETRY

P-31 NMR and potentiometric titration experiments allowed the complete microconstant system to be resolved for D-myo-inositol 1,2,6-tris(phosphate) [Ins(1,2,6)P-3] and DL-myo-inositol 1,2-bis(phosphate). For comparison, DL-myo-inositol 1-mono(phosphate) and DL-myo-inositol 2-mono(phosphate) were also considered. The studies have been performed in 0.1 RI tetraethylammonium perchlorate or tetrabutylammonium bromide solutions at 25 degrees C (media 1) and, in addition, for Ins(1,2,6)P-3 in a 0.2 M KCl medium at 37 degrees C (medium 2). The P-31 NMR titration curves of the polyphosphates indicate that the protonation process of a given phosphate group is complex and far from that of a simple monoester. The interactivity parameters derived from the microconstants support the conclusion that two cis phosphates interact Less than two trans phosphates. The influence of the presence of Kf on the microconstants determined in medium 2 can be explained by the competition between H+ and K+ for binding to the ligand. The distribution curves of the microspecies versus pH allow a direct determination of the protonation state of each phosphate group.