Intrinsic Ca2+ affinities of peptides: Application of the kinetic method to analogs of calcium-binding site III of rabbit skeletal troponin C

We extended the kinetic method to determine the intrinsic affinities of non volatile organic molecules with divalent metal ions and then applied the am ended method to determine the calcium affinities of peptide analogs of the calcium-binding site III of rabbit skeletal troponin C. Metal-bis(peptide) complexes of the composition ([H2Pi + H2Pii] - H + Ca)(+), where H2P is a n eutral peptide, were introduced into the gas phase by fast atom bombardment . The extended kinetic method recognizes that the dissociation characterist ics of a singly charged, bis(peptide) complexes of divalent metal ions are determined by not only the metal-ion affinity but also the proton affinitie s of the neutral and deprotonated peptides. The modified method requires on e to measure the relative abundances of [H2P - H + Ca](+), [H2P + H](+), an d [H2P - H](-) ions that form upon collisional activation of mixed peptide/ metal complexes, proton-bound peptide dimers, and deprotonated peptide dime rs, respectively. We found, by using the modified method, that the set of p eptides has a different affinity order than that in solution. Peptides with one aspartic acid have a higher intrinsic Ca2+ affinity than those with tw o aspartates. The location of the aspartic acid (Asp) residues at various p ositions also affects the Ca affinity. Those peptides with one Asp in the m iddle of the chain have higher Ca2+ affinities than those with Asp on the e nd because the former peptides offer greater polarizability to stabilize th e charge. Peptides with two Asp's located in close proximity have higher in trinsic calcium affinities than those with aspartates positioned further ap art. (C) 2000 American Society for Mass Spectrometry.