Analysis of Ca2+ binding with gramicidin double helices using subcritical fluid chromatography

Subcritical fluid chromatography (subFC) has been demonstrated as a useful technique to directly analyze gramicidin monomers, double-helical dimers, and Ca2+-bound dimers in various solvents. While the data obtained agrees with earlier findings by other methods, it also yields new information about the Ca2+-gramicidin system. For instance, upon the addition of Ca2+ to methanol, all of the ion-free dimeric forms disappeared and a new peak emerged that is ascribed to the previously characterized Ca2+ species 1′. In ethanol and pentanol, however, in addition to species 1′ a second more abundant new species was also observed for the first time using subFC. Further, the relative abundance of both species formed in ethanol and pentanol was shown to be stable over time and independent of either solvent used. This is in contrast to ion-free dimers, which vary greatly in abundance with these parameters. Additional subFC data also confirmed that a significant fraction of monomer was simultaneously consumed during the formation of the emerging species. As well, the findings illustrated that Ca2+ binding stabilizes the dimers to the point of eliminating and even reversing the tendency toward monomerization that is normally observed for gramicidin in ion-free polar solvents. Results indicate that subFC is an informative complimentary tool for readily monitoring the dynamic changes that occur upon Ca2+ addition to gramicidin in different environments.