Effect of buffer cations and of H3O+ on the charge states of native proteins. Significance to determinations of stability constants of protein complexes.
The progressive reduction of charge in charge states of non-denatured proteins (lysozyme, ubiquitin, and cytochrome c), observed with nanospray in the positive ion mode, when the buffer salt ammonium acetate is replaced by ethylammonium acetates (EtNH(3)Ac, Et(2)NH(2)Ac and Et(3)NHAc) is rationalized on the basis of the charge residue model (CRM). The charge states of the multiply protonated protein are shown to be controlled by the increasing gas-phase basicities, GB(B), of the bases(B) NH(3), EtNH(2), Et(2)NH and Et(3)N. Charge states derived from evaluated apparent gas-phase basicities GB(app) of the basic side-chains of the protein and the known GB(B) of the above bases are found to be in agreement with the experimentally observed charge states. This is a requirement of the CRM, because in this model the small positive ions (the buffer cations in the present case) at the surface of the electrospray droplets are the excess ions that provide the charge of the final small droplet that contains the protein molecule and on evaporation of the solvent transfer the charge to the protein. The observed charge states in the absence of buffer salts, i.e. pure water, are attributed to excess H(3)O(+) ions produced by the electrolysis process that attends electrospray. A proposed extended mechanism provides predictions of factors that determine the sensitivity for detection of the multiply protonated proteins. Consideration of restraints imposed by the CRM lead to some simple predictions for conditions that should be present to obtain accurate determinations by electrospray and nanospray of stability constants for the protein-complex equilibrium in aqueous solution.
Verkerk
Udo H
UH
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Peschke
Michael
M
Kebarle
Paul
P
D000818:Animals; D002021:Buffers; D002412:Cations / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D009113:Muramidase / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D055672:Static Electricity; D014452:Ubiquitins / Q000737:chemistry
D002021:Buffers; D002412:Cations; D046911:Macromolecular Substances; D011506:Proteins; D011522:Protons; D000644:Quaternary Ammonium Compounds; D014452:Ubiquitins; D045304:Cytochromes c; D009113:Muramidase