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(pho sphate) [Ins(1,2,6)P-3] and DL-myo-inositol 1,2-bis(phosphate). For co mparison, 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 sol utions at 25 degrees C (media 1) and, in addition, for Ins(1,2,6)P-3 i n a 0.2 M KCl medium at 37 degrees C (medium 2). The P-31 NMR titratio n curves of the polyphosphates indicate that the protonation process o f a given phosphate group is complex and far from that of a simple mon oester. The interactivity parameters derived from the microconstants s upport the conclusion that two cis phosphates interact Less than two t rans phosphates. The influence of the presence of Kf on the microconst ants determined in medium 2 can be explained by the competition betwee n 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.