Measuring dissociation constants of RNA and aminoglycoside antibiotics by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) has been used to determi ne the dissociation constants (K(D)s) and binding stoichiometry for tobramy cin and paromomycin with a 27-nucleotide RNA construct representing the A-s ite of the 16S ribosomal RNA. K-D values determined by holding the ligand c oncentration fixed are compared with K-D values derived by holding the RNA target concentration fixed. Additionally, the effect of solution conditions such as the amount of organic solvent present and the amount of salt prese nt in the solution on the K-D measurement is investigated. It is shown that the preferred method for determining dissociation constants using ESI-MS i s holding the RNA target concentration fixed below the expected K-D and tit rating the ligand. K-D measurements should also be carried out at as high a s possible salt concentration to minimize nonspecific binding due primarily to electrostatic interactions. For tobramycin, two nonequivalent binding s ites were found with K-D1 = 352 nM and K-D2 = 9 mu M. For paromomycin, ther e is only one binding site with K-D = 52 nM. (C) 2000 Academic Press.