A KINETIC METHOD TO EVALUATE THE 2-STATE CHARACTER OF SOLVENT-INDUCEDPROTEIN DENATURATION

We present a kinetic method to determine the concentration of native m olecules in protein folding transitions. It is based on the observatio n that frequently native protein molecules unfold slowly when transfer red to unfolding conditions, whereas folding intermediates unfold rapi dly. The fraction of native molecules in a folding transition can thus be determined by kinetic unfolding assays in a two-step procedure. Al iquots of the protein are first equilibrated at different concentratio ns of denaturant and then transferred to constant unfolding conditions to determine the amplitude of unfolding. This amplitude is a direct m easure for the concentration of native molecules in the sample. The tw o-state character of a solvent-induced unfolding transition can thus b e examined. When the fractional change of a spectral property in a tra nsition follows the decrease in the concentration of the native molecu les, as measured by the unfolding assays, then the presence of interme diates that differ from the unfolded protein in this property can be d efinitely excluded. This test complements the calorimetric test for in termediates in thermal unfolding transitions. By using this method, we show that the NaCl-induced folding transition of the reduced and carb oxymethylated form of a variant of ribonuclease Tl is well described b y the two-state approximation. In the unfolding of apo-alpha-lactalbum in, the measured profile for the native protein coincides with the flu orescence-detected transition, but not with the transition that is mon itored by amide circular dichroism. This confirms that a partially fol ded intermediate is present in the folding transition of apo-alpha-lac talbumin.