Use of fast protein size-exclusion liquid chromatography to study the unfolding of proteins which denature through the molten globule

Abstract

Fast protein size-exclusion liquid chromatography (SEC-FPLC) was used to study solvent-induced unfolding of six proteins. Two of them (sperm whale myoglobin and hen white lysozyme) denature on the simple N (native)<-->U (completely unfolded) scheme. The other four proteins [bovine and human alpha-lactalbumin, bovine carbonic anhydrase B (BCAB), and beta-lactamase from Staphylococcus aureus] denature through the molten globule (MG) state (i.e., on the N<-->MG<-->U denaturation scheme). We have shown that the permeation properties of the Superose 12 columns are practically independent of temperature, pH, and denaturants in wide concentration intervals. In the case of myoglobin and lysozyme denaturation at 4 degrees C (when the exchange between the native and unfolded states is slower than the characteristic time of chromatography), a bimodal distribution on molecular dimensions in the transition region was observed. This indicates that, under denaturant action, protein molecules can only be in one of the two states with different compactness. In other words, this shows that FPLC is one of the most direct approaches to establish the "all-or-none" mechanism of the equilibrium solvent-induced denaturation of globular proteins. The curves of guanidinium hydrochloride- (GdmHCl) or urea-induced unfolding (N<-->U or MG<-->U transitions) of a protein on a column (monitored either by the relative areas of two peaks or--for fast exchange--by the position of the average peak) coincide with those monitored by far-UV CD in solution. The Stokes radius values obtained with the use of FPLC for the molten globule states of BCAB (1.6 M GdmHCl in 0.1 M sodium phosphate, pH 6.8, and acid form at pH 3.6) and for the human alpha-lactalbumin molten globule (2.0 M GdmHCl in 0.1 M sodium phosphate, pH 6.8) coincide with those known from literature. Thus, it has been shown that fast protein size-exclusion liquid chromatography (FPLC) is an "inert" technique, i.e., it does not shift the equilibrium between N, MG, and U states and, therefore, can be used for qualitative and quantitative studies of protein denaturation.