On the mechanism of globular protein unfolding. Ribonuclease A

Ribonuclease A unfolding in urea and guanidinium hydrochloride solutions was studied by tritium labeling. With both denaturing agents, unfolding does not occur as a transition between two states, as generally accepted, but involves formation of a state in which the globule compactness is intermediate between those in the native and the completely unfolded states. With urea, the intermediate state is formed through gradual swelling of the native globule, with an increase in volume by 60%. The sizes of the hydrophilic and hydrophobic surfaces exposed to the solvent are approximately equal in this state. With guanidinium hydrochloride, the intermediate state is nearly as compact as the molten globule state (the increase in volume is 18%) and its formation resembles a phase transition of the first type. The polar surface exposed in the presence of guanidinium hydrochloride is less by a factor of five, which allows one to consider this state as a "dry molten globule," by contrast with a "damp molten globule" found in the presence of urea. The intermediate formation induced by guanidinium hydrochloride is preceded by an 8% decrease in the volume of the globule. Enzyme activity is completely inhibited at this step. In urea solutions, there is a correlation between activity and the accessible surface area of the globule, which confirms the two-step mechanism of denaturation. The unfolded state is achieved through swelling of the intermediate state with both denaturing agents.