Amino Acids Stabilizing Effect on Protein and Colloidal Dispersions

Abstract

Despite being used for decades as stabilizers, amino acids (AAs) remain mysterious components of many medical and biological formulations. Hypotheses on their role vary ranging from hydrotropic to protein-specific effects (stabilization against misfolding). Here, we deduce that AAs possess a new and broad colloidal property by finding that stabilizing effect of the AAs is comparable on dispersion of various proteins, plasmid DNA, and non-biological nanoparticles. The interactions among colloidal particles in dispersion are carefully evaluated by the second osmotic virial coefficient (B_22) and the potential of mean force. We propose a theoretical framework that explains the stabilization as the effect of weakly interacting small molecules with patchy nanoscale colloids. We validate it through quantitative comparison with experimental data by comparing equilibrium dissociation constants for AA/proteins obtained either by fitting the B22 data with this theory or experimentally. We find excellent quantitative agreement (e.g. proline/lysozyme 1.18 and 2.28 M, respectively) and indeed that the interactions are very weak. The theory presented implies that (i) charged AAs will be effective only for proteins of opposite charge; (ii) short peptides composed of n AAs will be as or more effective than n separate AAs; (iii) any small molecule weakly interacting with nanoscale colloids that increases the solvation of the surface will have a stabilizing effect. The experimental evidences corroborate all three predictions. Much like the ionic strength of the solution is commonly reported, our results imply that the same should be done for the small molecules, as they also affect fundamentally colloidal properties. As an example, we show that AAs vary the cloud point of a lysozyme solution by as much as 4 K.