The Effect of Buffers on Protein Conformational Stability

*To whom all correspondence should be addressed. uffers used to formulate proteins should not serve as substrates or inhibitors. They should exhibit little or no change in pH with temperature, show insignificant penetration through biological membranes, and have maximum buffer capacity at a pH where the protein exhibits optimal stability. In conformity with the proposition that “Nature designs the optimum molecules,” buffers should mimic the antidenaturant properties of nature exhibited by osmolytes (1–5) that are independent of the evolutionary history of the proteins (6, 7). Such properties may include preferential exclusion from the protein domain (8–11) and stabilization without changing the denaturation Gibbs energy ( Gd) (12). Conformational instability refers not only to unfolding, aggregation, or denaturation but also to subtle changes in localized protein domains and the alteration of enzyme catalytic properties (13) that may result from buffer-component binding, proton transfer, and metal or substrate binding effects directly or indirectly mediated by buffers or by buffers themselves acting as pseudosubstrates. Salts can affect protein conformation to the extent that the anions or cations of the salt could be potential buffer components. When the salt concentration is much larger than that of the buffer, the salt becomes the effective buffer in the reaction. The mechanisms or combinations thereof by which buffers may cause protein stabilization (or destabilization) are complex and not well understood. The problem is compounded by the inability to definitively differentiate between various protein stabilization mechanisms, the small free energies of stabilization of globular proteins (14–16), and a paucity of review manuscripts on this subject in the literature. The authors address some of these issues as they relate to buffers used in the formulation of proteins. The effect of buffers that may be used in the extraction, purification, dialysis, refolding, or assay of proteins on protein conformation is not discussed.