Unfoldness of the denatured state of proteins determines urea: Methylamine counteraction in terms of Gibbs free energy of stabilization.

In many tissues and organisms, large amount of urea gets accumulated to maintain osmotic balance. To evade the threatening impact of urea, living organisms accumulate methylamines, a class of osmolytes, in proportion of 2:1 (urea:methylamine). To understand underlying cause(s) for protein-specific counteraction behavior, thermodynamic stability (ΔG) of three disulfide free proteins (myoglobin, bovine cytochrome c and barstar) in the mixture of urea and methylamine has been estimated from guanidinium chloride-(GdmCl) driven denaturation curves. Using the experimentally measured values of ΔG obtained in the presence of individual methylamines and urea, we predicted the molar ratio of urea and a methylamine required for perfect compensation for each of the proteins. Interestingly, for all proteins studied, a similar ratio has been observed for perfect compensation. The predicted ratio for perfect compensation in terms of thermodynamic parameters was about 2:1 M ratio of urea to methylamine. Furthermore, a partial counteraction was observed in the myoglobin and barstar. However, for bovine cytochrome c, perfect compensation was observed in both GdmCl- and heat-driven denaturations. Our observations clearly suggest that the counteraction phenomenon depends on the extent of the unfolding of the denatured states of proteins.