19F NMR magnetization transfer between 5-FBAPTA and its complexes. An alternative means for measuring free Ca2+...

The 19F NMR Ca(2+)-indicator molecule 5,5'-difluoro-1,2-bis(o- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5-FBAPTA) was used in a procedure that enhances its range of applications to measuring free Ca2+ concentrations in buffer solutions and human erythrocytes. Even if the signal from the Ca-5-FBAPTA complex was not visible, the concentration of the complex could be calculated from saturation transfer spectra. This was demonstrated with well characterized buffer solutions in vitro and shown to also apply to concentrated haemolysates. The analysis required a precise estimate of the dissociation rate constant of the complex; this was found to be 295/s at 37 degrees C and the corresponding association rate constant was 4.1 x 10(8) L/mol/s. These values differ from those obtained previously in different buffer conditions and by two different NMR methods. A series of spectra were acquired from haemolysates containing 5-FBAPTA, in which saturating irradiation was applied at a frequency that was progressively off-set from the carrier frequency. Saturation transfer to the free 5-FBAPTA was seen from irradiation at frequencies different from that of Ca-5-FBAPTA, thus suggesting the presence of complexes with proteins.