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

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