Osmolyte mixtures have different effects than individual osmolytes on protein folding and functional activity

• Osmolyte mixtures have higher protein stabilizing ability than individuals.
• Individual osmolytes increase enzyme activity while mixtures cancel it out.
• Osmolyte mixtures induces poor protein refolding relative to individuals.

Osmolytes are small organic molecules accumulated by organisms under stress conditions to protect macromolecular structure and function. In the present study, we have investigated the effect of several binary osmolyte mixtures on the protein folding/stability and function of RNase-A. For this, we have measured ΔGDo (Gibbs free energy change at 25 °C) and specific activity of RNase-A mediated hydrolysis of cytidine 2′–3′ cyclic monophosphate in the presence and absence of individual and osmolyte mixtures. It was found that the osmolyte mixtures have different effect on protein stability and function than that of individual osmolytes. Refolding studies of RNase-A in the presence of osmolyte mixtures and individual osmolytes also revealed that osmolyte mixtures have a poor refolding efficiency relative to the individual osmolytes.