Protein interactions in concentrated ribonuclease solutions

To investigate the protein interactions involved in the crystallization pro cess of ribonuclease A, dynamic light scattering (DLS) and small angle X-ra y scattering experiments (SAXS) were performed on concentrated solutions. W hereas the translational diffusion coefficient obtained from DLS is sensiti ve to thermodynamic and hydrodynamic interactions and permits to calculate an interaction parameter, the shape of the SAXS curves is related to the ty pe of interaction (attractive or repulsive). We compared the effect of pH o n protein interactions in the case of two types of crystallizing agents: a mixture of salts (3 M sodium chloride plus 0.2 M ammonium sulfate) and an o rganic solvent (ethanol). The results show that in the presence of ethanol, as in low salt, protein interactions become more attractive as the pH incr eases from 4 to 8 and approaches the isoelectric point. In contrast, a reve rse effect is observed in high salt conditions: the strength of attractive interactions decreases as the pH increases. The range of the pH effect can be related to ionization of histidine residues, particularly those located in the active site of the protein. The present observations point out the i mportant role played by localized charges in crystallization conditions, wh atever the precipitating agent. (C) 1999 Elsevier Science B.V. All rights r eserved.