A novel view of pH titration in biomolecules

When individual titratable sites in a molecule interact with each other, th eir pH titration can be considerably more complex than that of an independe nt site described by the classical Henderson-Hasselbalch equation. We propo se a novel framework that decomposes any complex titration behavior into si mple standard components. The approach maps the set of N interacting sites in the molecule onto a set of N independent, noninteracting quasi-sites, ea ch characterized by a pK(a)' value. The titration curve of an individual si te in the molecule is a weighted sum of Henderson-Hasselbalch curves corres ponding to the quasi-sites. The total protonation curve is the unweighted s um of these Henderson-Hasselbalch curves. We show that pK(a)' values corres pond to deprotonation constants available from methods that can be used to assess total proton uptake or release, and establish their connection to pr otonation curves of individual residues obtained by NMR or infrared spectro scopy. The new framework is tested on a small molecule diethylenetriaminepe ntaacetate (DTPA) exhibiting nonmonotonic titration curves, where it gives an excellent fit to experimental data. We demonstrate that the titration cu rve of a site in a group of interacting sites can be accurately reconstruct ed, if titration curves of the other sites are known. The application of th e new framework to the protein rubredoxin demonstrates its usefulness in ca lculating and interpreting complicated titration curves.